Mass tag analysis for rare cells and cell free molecules

ABSTRACT

The invention generally relates to mass tag analysis for rare cells and cell free molecules. In certain embodiments, the invention provides an apparatus including an essentially non-absorbent membrane having at least one pore, a microwell operably associated with the essentially non-absorbent membrane, and an electric field generator. The apparatus may be configured such that an electric field produced by the electric field generator operably interacts with a sample in the microwell and expels a droplet of the sample through the at least one pore in the essentially non-absorbent membrane. In certain embodiments, apparatuses of the invention are used for detection, and optionally quantification, of a target analyte from a heterogeneous sample, such as a rare target analyte (e.g., rare cell) from a biological sample.

RELATED APPLICATION

The present application claims the benefit of and priority to U.S.provisional application Ser. No. 62/222,940, filed Sep. 24, 2015, thecontent of which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The invention generally relates to mass tag analysis for rare cells andcell free molecules.

BACKGROUND

Cellular analysis is important in medical applications such as, forexample, diagnosis of many diseases. The detection of rare moleculesthat are cell bound or included in the cell is also desirable. Themedical applications of cellular analysis require isolation of certaincells of interest, which typically represent only a small fraction of asample under analysis. For example, circulating tumor cells (“CTCs”) areof particular interest in the diagnosis of metastatic cancers. Inconventional methods, CTCs are isolated from whole blood by firstremoving red blood cells (RBCs) by lyses. In a 10 mL blood sample, a fewhundred CTCs are to be separated from about 800,000,000 white bloodcells (“WBCs”). Therefore, methods with high separation efficiency andcell recovery rates are necessary.

However, existing technologies are ineffective for detecting raremolecules from a sample. For example, the detection of rare moleculescannot be achieved by conventional affinity assays, which require anumber of molecular copies far above the numbers found for raremolecules. The detection of rare molecules can be achieved byconventional nucleic acid assays. However, the target nucleic acids mustbe subjected to one or more lengthy purification steps andamplifications that can take several days for analysis time.

Cell filtration for the separation of rare cells using a porous matrixis a useful method used to sort cells by size and, in most instances,such filtration methods allow for the extraction of cells followingseparation. However, the existing filtration methods are limited bycertain factors, which include, for example, the range of diameters thatin vitro cells have rather than a single diameter. Additionally, cellfiltration techniques yield only a few rare cells. The number of copiesof a rare molecule can be significant at only tens of thousands ofcopies per cell for proteins or a few copies per cell for a genemutation.

Rare cells can be analyzed down to the single cell level by aconventional scanning microscopy. However, even with automation of thescanning and analysis, the microscopy method can take 24 hours or morefor each sample to be scanned. Additionally, all the rare cells withmultiple images must be examined visually by the pathologist todetermine the significance of protein amounts measured.

Mass spectroscopy (MS) has several issues that keep MS from beingcompetitive with routine affinity reaction systems. The noted problemsare inability to separate markers of interest from sample interference(matrix over lapping peaks), loss of sensitivity due to background inclinical sample (picomolar (pM) reduced to nanomolar (nM)), theinability to work with small nL sample volumes as samples less than 1microliters (μl) are inefficiently captured for ionization andinefficiently isolated from interfering peaks in complex samples such asblood. In addition, MS often is not able to detect certain masses due tocompetition with other molecules of the same mass being ionized. Theseissues typically cause problems and provide false results.

SUMMARY

The invention recognizes that when mass spectral analysis is employed incarrying out rare target detection, it is important to avoid dilution ofthe detection liquid because dilution substantially reduces sensitivityof detection. Cells or capture particles in a detection liquid should beindividually detected because each has a unique nature. Accordingly, theinvention provides methods and apparatuses that provide for release ofprecise small amounts of detection liquid from a membrane and fordelivery of liquid droplets into a mass spectrometer while avoidingdilution of the detection liquid.

Aspects of the invention are accomplished with an apparatus thatincludes an essentially non-absorbent membrane (non-bibulous membrane)including at least one pore, a microwell operably associated with themembrane, and an electric field generator operably associated with themembrane. A heterogeneous sample (e.g., a blood sample) is introduced toat least the microwell, the membrane, or both of the apparatus. Aplurality of affinity agents are introduced to the sample. Each of theplurality of affinity agents includes a first molecule. The plurality ofaffinity agents specifically bind the target analyte in the sample.Unbound affinity agents are removed (e.g., by washing). One or moreadditional molecules are introduced to the sample. The one or moreadditional molecules interact with the first molecule to form a massspectrometry label. A voltage is provided to the sample via the electricfield generator in order to release a droplet through the at least onepore. The droplet includes a portion of the sample and the massspectrometry label. The droplet is analyzed for presence of the massspectrometry label, for example by mass spectrometry analysis of anionized mass spectrometry label. The presence of the mass spectrometrylabel indicates presence of the target analyte in the sample. In certainembodiments, methods of the invention may additionally involvequantifying the target analyte in the sample by quantifying an amount ofmass spectrometry label analyzed.

Generally, the at least one pore includes a proximal opening, a distalopening, and walls. Many different orientations of the pore are withinthe scope of the invention. For example, the walls of the at least onepore may oriented to be 90 degrees with respect to the proximal anddistal openings. In another embodiment, the walls of the at least onepore taper from the proximal opening toward the distal opening. Inanother embodiment, the walls of the at least one pore taper from thedistal opening toward the proximal opening.

In certain embodiments, the essentially non-absorbent membrane includesa plurality of pores. The plurality of pores may have the samedimensions. Alternatively, the plurality of pores may have differentdimensions. In such embodiments, the apparatus is configured to generatean electric field from the electric field generator that produces adroplet from only one of the plurality of pores.

The apparatus may further include a mass spectrometer. The massspectrometer may be a bench-top mass spectrometer or a miniature massspectrometer, such as described for example in Gao et al. (Z. Anal. 15Chem. 2006, 78, 5994-6002), Gao et al. (Anal. Chem., 80:7198-7205,2008), Hou et al. (Anal. Chem., 83:1857-1861, 2011), Sokol et al. (Int.J. Mass Spectrom., 2011, 306, 187-195), Xu et al. (JALA, 2010, 15,433-439); Ouyang et al. (Anal. Chem., 2009, 81, 2421-2425); Ouyang etal. (Ann. Rev. Anal. Chem., 2009, 2, 187-25 214); Sanders et al. (Euro.J. Mass Spectrom., 2009, 16, 11-20); Gao et al. (Anal. Chem., 2006,78(17), 5994-6002); Mulligan et al. (Chem.Com., 2006, 1709-1711); andFico et al. (Anal. Chem., 2007, 79, 8076-8082).), the content of each ofwhich is incorporated herein by reference in its entirety.

In certain embodiments, the apparatus is configured such that theelectric field generator inductively imparts the electric field to thesample in the microwell, such as described for example in U.S. Pat. No.9,184,036, the content of which is incorporated by reference herein inits entirety.

In certain embodiments, the first molecule is a mass spectrometry labelprecursor. In such embodiments, the one or more additional molecules isan alteration agent that interacts with the mass spectrometry labelprecursor to form the mass spectrometry label.

In other embodiments, the first molecule is an alteration agent. In suchembodiments, the one or more additional molecules is a mass spectrometryprecursor label that interacts with the alteration agent to form themass spectrometry label.

In certain embodiments, the first molecule is a mass spectrometry labelprecursor, and the one or more additional molecules are first and secondalteration agents that interact with the mass spectrometry labelprecursor to form the mass spectrometry label.

The affinity agent may be a particulate or a non-particulate. The targetanalyte may be a rare cell and the heterogeneous sample may be aheterogeneous biological sample.

Some exemplary methods of the invention are directed to methods ofreleasing liquid from an essentially non-absorbent membrane including atleast one pore. The essentially non-absorbent membrane may be associatedwith a microwell that is capable of holding liquid. An intersection ofthe at least one pore and at least one surface of the essentiallynon-absorbent membrane is at an angle of about 30° to about 150°. Themethod may involve exposing the liquid on the essentially non-absorbentmembrane to an electrical field to release one or more droplets of theliquid through the at least one pore of the essentially non-absorbentmembrane.

Other exemplary methods involve detecting one or more differentpopulations of target rare molecules in a sample suspected of containingthe one or more different populations of rare molecules and non-raremolecules. A sample (typically in liquid form), may be contacted to anapparatus that involves a microwell and an essentially non-absorbentmembrane having at least one pore. The intersection of the at least onepore and at least one surface of the essentially non-absorbent membraneis at an angle of about 30° to about 150°. The sample may be incubatedwith, for each different population of target rare molecules, anaffinity agent that includes a binding partner that is specific for andbinds to a target rare molecule of one of the populations of the targetrare molecules. The affinity agent includes a mass spectrometry labelprecursor or a first alteration agent. The affinity agent may benon-particulate or particulate. The first alteration agent eitherfacilitates the formation of a mass spectrometry label from the massspectrometry label precursor or releases an entity that includes themass spectrometry label precursor from the affinity agent. If the firstalteration agent does not facilitate the formation of a massspectrometry label from the mass spectrometry label precursor, thesample is subjected to a second alteration agent that facilitates theformation of a mass spectrometry label from the mass spectrometry labelprecursor. The mass spectrometry label corresponds to one of thepopulations of target rare molecules. The sample on the essentiallynon-absorbent membrane is exposed to an electrical field to release oneor more droplets of the sample through the at least one pore of theessentially non-absorbent membrane. The droplets are subjected to massspectrometry analysis to determine the presence and/or amount of eachdifferent mass spectrometry label. The presence and/or amount of eachdifferent mass spectrometry label may be correlated to the presenceand/or amount of each different population of target rare molecules inthe sample for each microwell.

In other aspects, the invention provides sample analysis methods thatinvolve introducing a sample suspected of comprising a target analyte toa membrane that includes a pore (e.g., an essentially non-absorbentmembrane but optionally an absorbent membrane). One or more reagents areintroduced to the sample on the membrane to generate a mass spectrometrylabel associated with target analyte if present in the sample. Anelectric field is applied to the membrane to thereby generate one ormore droplets of the sample that are expelled from the pore of themembrane and are introduced into a mass spectrometer. A presence of thetarget analyte is detected via the mass spectrometer by detecting apresence of the mass spectrometry label. A portion of the sampleassociated with the pore of the membrane is then extracted from themembrane if the target analyte is present based on results from thedetecting step, and the extracted portion of the sample is analyzed. Themethods of the invention may further involve quantifying the targetanalyte in the sample by quantifying an amount of mass spectrometrylabel analyzed.

The introduction of one or more reagents to the sample on the membranemay involve introducing to the sample a plurality of affinity agentsthat each include a first molecule, wherein the plurality of affinityagents specifically bind the target analyte in the sample, removingunbound affinity agents, and introducing one or more additionalmolecules to the sample, wherein the one or more additional moleculesinteract with the first molecule to form a mass spectrometry label.

In certain embodiments, the first molecule is a mass spectrometry labelprecursor. In such embodiments, the one or more additional molecules isan alteration agent that interacts with the mass spectrometry labelprecursor to form the mass spectrometry label.

In other embodiments, the first molecule is an alteration agent. In suchembodiments, the one or more additional molecules is a mass spectrometryprecursor label that interacts with the alteration agent to form themass spectrometry label.

In other embodiments, the first molecule is a mass spectrometry labelprecursor, and the one or more additional molecules are first and secondalteration agents that interact with the mass spectrometry labelprecursor to form the mass spectrometry label.

In all of these embodiments, the affinity agent may be a particulate ora non-particulate. In exemplary embodiments, the target analyte is arare cell and the sample is a heterogeneous biological sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C are schematics depicting examples of apparatuses of theinvention having different pore orientations.

FIG. 2 is a schematic depicting another example of an apparatus inaccordance with the invention.

FIG. 3 is a schematic depicting an example of an apparatus and method ofthe invention for releasing a liquid droplet from the apparatus shown inFIG. 1 , which droplet enters an intake of a mass spectrometer.

FIG. 4 is a schematic depicting an example of an apparatus and method ofthe invention having more than one pore. A droplet released from one ofthe pores enters an intake of a mass spectrometer.

FIG. 5 depicts the essentially non-absorbent membrane of the apparatusof FIG. 4 in which an area on the essentially non-absorbent membrane isidentified for further analysis.

FIG. 6 is a schematic depicting an example of a microwell array.

FIG. 7 is a schematic depicting an example of an apparatus including anarray and an electric field generator.

FIGS. 8A-C are a set of mass spectra showing FC-2 peptide sprayeddirectly from an apparatus of the invention. The set of mass spectra areMS/MS spectra for m/z 412 generated from the peptide FC-2 at variousconcentrations.

FIGS. 9A-C are a set of mass spectra showing FC-2 peptide sprayeddirectly from an apparatus of the invention. The set of mass spectra areMS/MS spectra for m/z 412 generated from the peptide FC-2 at variousconcentrations.

FIG. 10 is a photograph of a spray plume from an apparatus of theinvention that is directed into an MS inlet.

FIG. 11 shows a schematic showing a spray device for generating anddirecting a DESI-active spray.

FIG. 12 shows a schematic showing an embodiment of a low temperatureplasma (LTP) probe.

FIG. 13 shows a schematic of a liquid for generating ions being fed to apiece of paper for generation of an ion beam.

DETAILED DESCRIPTION

The invention generally relates to methods and apparatus for releasingliquid from a membrane especially in the area of analysis of smallamounts (on the microliter (μL) scale or less) of liquids that containonly a few molecules (on the femtomolar (fM) scale or less). In someaspects, the invention relates to methods, apparatuses and kits fordetecting one or more different populations of rare molecules in abiological sample (e.g., blood sample) suspected of containing the oneor more different populations of rare molecules and non-rare molecules.In some aspects, the invention relates to methods and kits for detectingone or more different populations of rare molecules that are freelycirculating in a biological sample (e.g., blood). In other aspects, theinvention relates to methods and kits for detecting one or moredifferent populations of rare molecules that are associated with rarecells in a biological sample (e.g., blood sample) suspected ofcontaining the one or more different populations of rare cells andnon-rare cells.

Methods of the invention for detecting one or more different populationsof target rare molecules in a sample may involve enhancing theconcentration of the one or more different populations of target raremolecules over that of the non-rare molecules. A concentrated sample isformed and is incubated with, for each different population of targetrare molecules, an affinity agent that comprises a binding partner thatis specific for and binds to a target rare molecule of one of thepopulations of the target rare molecules. The affinity agent may benon-particulate or particulate. The affinity agent comprises a massspectrometry (MS) label precursor or a first alteration agent, whicheither facilitates the formation of an MS label from the MS labelprecursor or releases an entity that comprises the MS label precursorfrom the affinity agent. The MS label corresponds to one of thepopulations of target rare molecules. A retentate and a filtrate areformed by contacting the incubated sample with a porous matrix. One orboth of the retentate and the filtrate are subjected to a secondalteration agent that facilitates the formation of a MS label from theMS label precursor from the affinity agent if the first alteration agentdoes not facilitate the formation of a MS label from the MS labelprecursor. One or both of the retentate and the filtrate are subjectedto MS analysis to determine the presence and/or amount of each differentMS label. The presence and/or amount of each different MS label arerelated to the presence and/or amount of each different population oftarget rare molecules in the sample.

In another example, a ratio of rare cells to non-rare cells in a bloodsample suspected of containing rare cells and non-rare cells isincreased. A treated blood sample is prepared by providing incombination the blood sample, a platelet deactivation agent, afibrin-formation-arresting agent and fibrin in an amount sufficient tocause a predetermined level of agglutination of the rare cells. Thetreated blood sample is then contacted with a porous matrix such thatagglutinated rare cells are preferentially retained on the porousmatrix.

In another example, methods of separating rare cells with intact nucleicacids from non-rare cells in a sample comprising the rare cells andnon-rare cells are employed. The sample is combined with an aqueousmedium, and the combination is held for a period of time and at atemperature for selectively releasing nucleic acids from the non-rarecells but not from the rare cells. The sample is subjected to filtrationto separate rare cells from non-rare cells.

General Discussion

The apparatuses described herein permit formation of ionized dropletsfrom a small quantity of liquid that is retained on an essentiallynon-absorbent membrane and further permits the subsequent release of thedroplets from the essentially non-absorbent membrane through at leastone pore of the essentially non-absorbent membrane (non-bibulousmembrane).

An example of an apparatus of the invention is depicted in FIG. 1 .Apparatus 10 comprises circular wall 12 having microwell 14 andessentially non-absorbent membrane 16 with at least one pore 18. Pore 18and essentially non-absorbent membrane 16 intersect at point 20 at anangle of 90°. The pore acts to facilitate generation and release ofdroplets (e.g., liquid droplets) from the apparatus 10.

Another example of an apparatus of the invention is depicted in FIG. 2 .Apparatus 30 comprises circular wall 32 having microwell 34 andessentially non-absorbent membrane 36 with at least one pore 38. Uppersurface 36 a of membrane 36 and inner surface 38 a of pore 38 intersectat point 40 at an angle of 90° and lower surface 36 b of membrane 36 andinner surface 38 a of pore 38 intersect at point 42 at an angle of 90°.The pore acts to facilitate generation and release of droplets (e.g.,liquid droplets) from the apparatus 30.

FIG. 3 depicts an example of releasing a droplet (e.g., liquid droplet)from an apparatus as described in FIG. 1 . Liquid 24 is contained inmicrowell 14 and does not have sufficient volume to pass through pore 18of essentially non-absorbent membrane 16. Electric field generator 20 isactivated by wire 20 a to produce an electric field having sufficientintensity to result in the release of droplet 24 a from essentiallynon-absorbent membrane 16 through pore 18. Droplet 24 a is collected ininlet 26 of mass spectrometer 28.

Mass spectrometer 28 can be any type of mass spectrometer known in theart, such as a bench-top mass spectrometer or a miniature massspectrometer. An exemplary miniature mass spectrometer is described, forexample in Gao et al. (Z. Anal. Chem. 2006, 78, 5994-6002), the contentof which is incorporated by reference herein in its entirety Incomparison with the pumping system used for lab-scale instruments withthousands watts of power, miniature mass spectrometers generally havesmaller pumping systems, such as a 18 W pumping system with only a 5L/min (0.3 m3/hr) diaphragm pump and a 11 L/s turbo pump for the systemdescribed in Gao et al. Other exemplary miniature mass spectrometers aredescribed for example in Gao et al. (Anal. Chem., 80:7198-7205, 2008),Hou et al. (Anal. Chem., 83:1857-1861, 2011), and Sokol et al. (Int. J.Mass Spectrom., 2011, 306, 187-195), the content of each of which isincorporated herein by reference in its entirety. Miniature massspectrometers are also described, for example in Xu et al. (JALA, 2010,15, 433-439); Ouyang et al. (Anal. Chem., 2009, 81, 2421-2425); Ouyanget al. (Ann. Rev. Anal. Chem., 2009, 2, 187-214); Sanders et al. (Euro.J. Mass Spectrom., 2009, 16, 11-20); Gao et al. (Anal. Chem., 2006,78(17), 5994-6002); Mulligan et al. (Chem.Com., 2006, 1709-1711); andFico et al. (Anal. Chem., 2007, 79, 8076-8082).), the content of each ofwhich is incorporated herein by reference in its entirety.

FIG. 4 depicts another example of an apparatus of the invention thatincludes an array of pores. Liquid 64 is contained in microwell 54 ofapparatus 50 and does not have sufficient volume to pass through pores58 a-58 d of essentially non-absorbent membrane 56. Microwell 54 hascircular wall 56. Electric field generator 60 is activated to produce anelectric field having sufficient intensity to result in the release ofdroplet 64 a from essentially non-absorbent membrane 56 only throughindividual pore 58 b. Droplet 64 a is collected in inlet 66 of massspectrometer 68. In this example, the dimensions of the electric fieldgenerator are selected to apply an electrical field precisely to asingle pore or to a subset of pores. Thus, the electrical fieldgenerator is designed accordingly so that the electrical field generatorincludes at least a portion (such as, e.g., a tip, wire, needle, cone,rectangle, or sphere) that permits such an application. In this example,the dimensions of the electrical field generator at the point ofapplication of the electrical field should be about the size of the poreor the subset of pores to which selective application of the electricalfield is desired. Thus, the dimensions of the electrical field generatorat the point of application of the electrical field should be no greaterthan about 200% and no less than about 50%, or no greater than about150% and no less than about 25%, or no greater than about 100% and noless than about 50%, or no greater than about 50% and no less than about25%, of the size of the pore or the subset of pores. Furthermore, theinlet of a mass spectrometer should be aligned with the electrical fieldgenerator. In some examples, the inlet of the mass spectrometer hasdimensions that correspond with that of the electrical field generatorat the point of application of the electrical field.

Referring to FIGS. 4 and 5 , an MS label in droplet 64 a is identifiedas a result of MS analysis and a corresponding area 59 on essentiallynon-absorbent membrane 56 is selected for further analysis. Liquid orparticle (including cell) is removed from area 59 by suction, punchingout, dissection, or extraction, for example, or a combination of two ormore of the above.

In the apparatuses described above, the essentially non-absorbentmembrane may be a flat surface that is essentially or completelyimpermeable to the liquid. The essentially non-absorbent membraneincludes at least one pore, and in certain embodiments more than onepore (e.g., an array of pores). The at least one pore has a fixedorientation within the essentially non-absorbent membrane. That fixedorientation may be described with respect to how the walls of the poreintersect the surface of the essentially non-absorbent membrane. Forexample, the pore can have vertical walls such that the walls of thepore intersect the surfaces (top surface that faces the microwell(proximal surface) and bottom surfaces that faces the mass spectrometer(distal surface)) of the essentially non-absorbent membrane at 90degrees. Such an orientation of the pore is shown in FIG. 1A.

Other orientations are possible and the skilled artisan will appreciatethat the invention is not limited to a specific orientation of the pore.For example, the walls of the pore can intersect the surfaces of theessentially non-absorbent membrane at an angle at the intersection ofthe two surfaces of about 30° to about 150°. That allows for the pore totaper from the proximal surface toward the distal surface as shown inFIG. 1B (i.e., the pore is dimensioned to become more narrow).Alternatively, the pore can taper from the distal surface to theproximal surface FIG. 1C (i.e., the pore is dimensioned to becomebroader). In some examples where the essentially non-absorbent membranecomprises more than one pore, the angle at the intersection has a highdegree of precision (less than 1 degree of variability) from one pore toanother, i.e., the pores all have the same dimensions. Thus, in thisexample, an angle of a pore and a surface of the essentiallynon-absorbent membrane does not differ from an angle of another pore bymore than 1°. In other embodiments, the some or all of the pores havedifferent dimensions from each other.

The term “intersection” means the point or series of points where twosurfaces touch one another. In some examples where the essentiallynon-absorbent membrane comprises more than one pore, an angle of one ofthe pores and a surface of the essentially non-absorbent membrane doesnot differ from an angle of another pore by more than 1°, or by morethan 0.5°, or by 0.2°, or by more than 0.1°, or by more 0.05°, or bymore than 0.01°, or by more than 0.005°, or by more than or by more than0.001°, for example.

In some examples, the liquid on the essentially non-absorbent membraneis exposed to an electrical field to cause release of one or more liquiddroplets from the essentially non-absorbent membrane. The electric fieldcan also cause ionization of molecules within the droplets. Theessentially non-absorbent membrane is also associated with an electricalfield generator. Activation of the electrical field generator producesan electrical field, which causes liquid to more through the pore andform a liquid droplets that is released from the membrane through the atleast one pore into, for example, an inlet of a mass spectrometer.

As mentioned above, the essentially non-absorbent membrane is associatedwith a microwell capable of holding liquid. The phrase “associated with”means that the essentially non-absorbent membrane and the microwell mayform a single unit in which the essentially non-absorbent membrane maybe on the bottom of the microwell or on the top of the microwell.

The liquid may be the sample or a liquid that contains an MS label. Theliquid may also be the MS label that is introduced to the sample. Insome examples, the liquid comprises a solvent such as, for example, aspray solvent employed in electrospray mass spectroscopy. In someexamples, solvents for electrospray ionization include, but are notlimited to, polar organic compounds such as, e.g., alcohols (e.g.,methanol, ethanol and propanol), acetonitrile, dichloromethane,dichloroethane, tetrahydrofuran, dimethylformamide, dimethylsulphoxide,and nitromethane; non-polar organic compounds such as, e.g., hexane,toluene, cyclohexane; and water, for example, or combinations of two ormore thereof. Optionally, the solvents may contain one or more of anacid or a base as a modifier (such as, volatile salts and buffer, e.g.,ammonium acetate, ammonium biocarbonate, volatile acids such as formicacid, acetic acids or trifluoroacetic acid, heptafluorobutyric acid,sodium dodecyl sulphate, ethylenediamine tetraacetic acid, andnon-volatile salts or buffers such as, e.g., chlorides and phosphates ofsodium and potassium, for example.

The membrane is essentially non-absorbent, which means that the membraneis essentially incapable of absorbing liquid (non-bibulous). In someexamples, the amount of liquid absorbed by the essentially non-absorbentmembrane is less than about 2% (by volume), or less than about 1%, orless than about 0.5%, or less than about 0.1%, or less than about 0.01%,or 0%. The essentially non-absorbent membrane may be non-fibrous, whichmeans that the membrane is at least 95% free of fibers, or at least 99%free of fibers, or at least 99.5%, or at least 99.9% free of fibers, or100% free of fibers.

The essentially non-absorbent membrane can be a solid, non-flexiblematerial, which is impermeable to liquid (except through one or morepores of the membrane). The essentially non-absorbent membrane may becomprised of an organic or inorganic material or a water insolublematerial. The shape of the essentially non-absorbent membrane isdependent on one or more of the nature of a holder or retainer for theessentially non-absorbent membrane, the nature and shape of the pore,the angle of the pore and the essentially non-absorbent membrane, thenature of the micro well, the nature of the charge generation, and thenature of a mass label, for example. In some examples the shape of theessentially non-absorbent membrane is circular, oval, rectangular,square, hexagonal, planar or flat surface (e.g., strip, disk, film,membrane, and plate), for example. In some examples the essentiallynon-absorbent membrane is rigid or non-flexible, which means that theessentially non-absorbent membrane may be flexed no more than about 1°,or no more than about 0.5°, or no more than about 0.1° from a plane ofthe essentially non-absorbent membrane.

The essentially non-absorbent membrane may be fabricated from a widevariety of materials, which may be naturally occurring or synthetic,polymeric or non-polymeric. Examples, by way of illustration and notlimitation, of such materials for fabricating an essentiallynon-absorbent membrane include plastics such as, for example,polycarbonate, poly (vinyl chloride), polyacrylamide, polyacrylate,polyethylene, polypropylene, poly(4-methylbutene), polystyrene,polymethacrylate, poly(ethylene terephthalate), nylon, poly(vinylbutyrate), poly(chlorotrifluoroethylene), poly(vinyl butyrate),polyimide, polyurethane, and paraylene; silanes; silicon; siliconnitride; graphite; ceramic material (such, e.g., as alumina, zirconia,PZT, silicon carbide, aluminum nitride); metallic material (such as,e.g., gold, tantalum, tungsten, platinum, and aluminum); glass (such as,e.g., borosilicate, soda lime glass, and PYREX (low-thermal-expansionborosilicate glass, Corning Incorporated)); and bioresorbable polymers(such as, e.g., poly-lactic acid, polycaprolactone and polyglycoicacid); for example, either used by themselves or in conjunction with oneanother and/or with other materials. The material for fabrication of theessentially non-absorbent membrane does not include fibrous materialssuch as cellulose (including paper), nitrocellulose, cellulose acetate,rayon, diacetate, lignins, mineral fibers, fibrous proteins, collagens,synthetic fibers (such as nylons, dacron, olefin, acrylic, polyesterfibers, for example) or, other fibrous materials (glass fiber, metallicfibers), which are bibulous and/or permeable and, thus, are not inaccordance with the principles described herein.

The essentially non-absorbent membrane for each microwell comprises atleast one pore. The essentially non-absorbent membrane can include morethan one pore, such as about 2,000,000 pores per square centimeter(cm²). In some examples the number of pores of the essentiallynon-absorbent membrane per cm² is 1 to about 2,000,000, or 1 to about1,000,000, or 1 to about 500,000, or 1 to about 200,000, or 1 to about100,000, or 1 to about 50,000, or 1 to about 25,000, or 1 to about10,000, or 1 to about 5,000, or 1 to about 1,000, or 1 to about 500, or1 to about 200, or 1 to about 100, or 1 to about 50, or 1 to about 20,or 1 to about 10, or 2 to about 500,000, or 2 to about 200,000, or 2 toabout 100,000, or 2 to about 50,000, or 2 to about 25,000, or 2 to about10,000, or 2 to about 5,000, or 2 to about 1,000, or 2 to about 500, or2 to about 200, or 2 to about 100, or 2 to about 50, or 2 to about 20,or 2 to about 10, or 5 to about 200,000, or 5 to about 100,000, or 5 toabout 50,000, or 5 to about 25,000, or 5 to about 10,000, or 5 to about5,000, or 5 to about 1,000, or 5 to about 500, or 5 to about 200, or 5to about 100, or 5 to about 50, or 5 to about 20, or 5 to about 10, forexample. The density of pores in the essentially non-absorbent membraneis about 1% to about 20%, or about 1% to about 10%, or about 1% to about5%, or about 5% to about 20%, or about 5% to about 10%, for example, ofthe surface area of the essentially non-absorbent membrane. In someexamples, the size of the pores of an essentially non-absorbent membraneis that which is sufficient to preferentially retain liquid whileallowing the passage of liquid droplets formed in as described herein.The size of the pores of the essentially non-absorbent membrane isdependent on the nature of the liquid, the size of the cell, the size ofthe capture particle, the size of mass label, the size of an analyte,the size of label particles, the size of non-rare molecules, and thesize of non-rare cells, for example. In some examples the average sizeof the pores of the essentially non-absorbent membrane is about 0.1 toabout 20 microns, or about 0.1 to about 5 microns, or about 0.1 to about1 micron, or about 1 to about 20 microns, or about 1 to about 5 microns,or about 1 to about 2 microns, or about 5 to about 20 microns, or about5 to about 10 microns, for example.

As mentioned above, the intersection of a top and/or a bottom surface ofthe essentially non-absorbent membrane and an inner wall of a pore hasan angle of about 30° to about 150°, or about 30° to about 125°, orabout 30° to about 110°, or about 30° to about 100°, or about 30° toabout 95°, or about 30° to about 90°, or about 45° to about 150°, orabout 60° to about 150°, or about 75° to about 150°, or about 80° toabout 150°, or about 85° to about 150°, or about 90° to about 150°, orabout 45° to about 125°, or about 60° to about 110°, or about 70° toabout 100°, or about 80° to about 100°, or about 85° to about 95°, orabout 90°, for example. The intersection of the surfaces depends on theshape of each of the surfaces such as, for example, the pore, and may belinear, circular, oval, hexagonal, square, or rectangular, for example,or a combination thereof.

The above characteristics of membranes allow a high level of precisionin an amount of liquid released as droplets from the membrane. Thevariation (CV) in an amount of liquid in droplets may be no more thanabout 1% (volume/volume), or no more than about 0.5%, or no more thanabout 0.1%, for example. Furthermore, the time at which the massdesorption from the solvent occurs (desorption time) is less variable,i.e., variable by no more than about 500 millisecond(s) (msec), or nomore than about 400 msec, or no more than about 300 msec, or no morethan about 200 msec, or no more than about 100 msec, or no more thanabout 50 msec, or no more than about 10 msec, thereby making thetrapping of ions much more facile at short time, thus permitting smallerspray volumes in comparison to known methods. Desorption time isdecreased further for rigid essentially non-absorbent membranes. Inaddition, shorter desorption times on the order of 50 msec may berealized where the essentially non-absorbent membrane comprises morethan one pore and the angle for one pore at a surface of the essentiallynon-absorbent membrane does not differ from an angle for another pore bymore than 0.5°. The precision obtained with apparatuses described hereinallows for highly quantitative results. The phrase “mass desorption”refers to the separation of mass label ions from solvent molecules.

Microwells and membranes with pores may be fabricated by, for example,microelectromechanical (MEMS) technology, metal oxide semiconductor(CMOS) technology, micro-manufacturing processes for producingmicrosieves, laser technology, irradiation, molding, and micromachining,for example, or a combination thereof.

As mentioned above, the emission of analyte (or mass tag) containingcharged droplets and analyte ions from pores of the essentiallynon-absorbent membrane (non-bibulous membrane) is accomplished by thegeneration of an electric field in the vicinity of the membrane. Theelectric field is established by providing an electrical potential ofabout 1 kilovolt (kV) to about 10 kilovolts (kV), or about 1 kV to about5 kV, or about 2 kV to about 10 kV, or about 5 kV to about 10 kV, orabout 6.0 to 6.5 kV to a conductive element (hereafter referred to asthe electric field generator) located 0.05 mm up to 20 mm distant fromthe essentially non-absorbent membrane. The apparatus is typicallypositioned a distance of 0.01 mm to 5 mm from the inlet capillary of amass spectrometer, which may be held at a potential of −300 V up to +300V.

The nature and intensity of the electric field is dependent on one ormore of the following: the nature of the liquid, the pore size, theamount of spray liquid, the distance between the membrane and theelectric field generator, the distance between the membrane and theinlet of the mass spectrometer, and the potentials applied to theelectric field generator and the inlet of the mass spectrometer. In somecases the electrical potential is supplied continuously via a highvoltage source in order to generate a continuous spray from themembrane. In other cases, the electrical potential is supplied bycompressing or decompressing a piezo-electric device (such as ananti-static gun) that is connected to the electric field generator.Furthermore, discrete emission of charged droplets and analytes from themembrane may be accomplished by providing one, or a series of electricalpulses in the range of 1 kV to about 15 kV, to the electric fieldgenerator for a duration from as little as 0.5 ms per individual pulseto as much as 2 minutes per individual pulse.

The volume of liquid expelled through the pore or the subset of pores isdependent on the volume of the samples, the size of the pore, nature ofanalysis, size of the well, the number of pores in a well, the number ofwells in the generated filed, the number of pores in the generatedfield, the pore size, the pore angle, and the rigidity of the membrane,for example. In some examples, the volume of liquid expelled is about 1fL to about 1 μL, or about 1 nL to about 1 μL, for example.

In some examples, an electrical field generator is associated with theessentially non-absorbent membrane and is activated to produce anelectrical field. In some examples the electrical field generator is anelectrical grid line integral with a support for the essentiallynon-absorbent membrane. In some examples the electrical field generatoris an electrical grid separate from the essentially non-absorbentmembrane and is disposed for movement to and from the essentiallynon-absorbent membrane. In some examples one or both of the electricalfield generator and the essentially non-absorbent membrane are attachedto a robotic arm that is capable of movement to bring the electricalfield generator into disposition with respect to the essentiallynon-absorbent membrane to permit activation of the electrical fieldgenerator to selectively induce droplet formation on an area of theessentially non-absorbent membrane or on a particular essentiallynon-absorbent membrane or group of essentially non-absorbent membraneswhere the essentially non-absorbent membrane may be part of an array ofessentially non-absorbent membranes as discussed below.

In some examples of the electrical field generator is a line, a plate,an ion stream or combinations thereof. Application of, for example, anelectrical potential, to the electrical field generator results inactivation of the electrical field generator. An ion stream may beproduced by different means including, but not limited to the generationof a plasma by dielectric barrier discharge, the application of analternating electrical potential to a suitable conductive element, theapplication of a static electrical potential to a suitable conductiveelement, or the compression of a piezoelectric material which isconnected to a suitable conductive element. In each case, the suitableconductive element is composed of an electrical conductive material ofsuitable geometry such that the electric field strength (uponapplication of electrical potential) is of sufficient magnitude to causeelectrical breakdown of the surrounding medium. In some cases thissuitable conductive element may be a wire, a protrusion or series ofprotrusions, a plate, a grid or mesh, a pointed rod, or a roughenedsurface. An ion stream may also be produced by electrospraying asuitable liquid. The generated ion stream is directed at one side of thenon-bibulous membrane while the opposite side of the membrane ispositioned near the inlet of a mass spectrometer as describedpreviously. The ion stream may be directed by, but is not limited to,positioning the ion stream generator in an appropriate manner such thatthe ion stream travels toward and impinges on the non-bibulous membrane,providing suitable electrical potentials to a series of conductiveelectrodes to electrostatically direct ions toward the membrane, orthrough the use of pneumatic forces—such as a flowing gas—to carry theion stream towards the membrane. Inductively charging and inductiveionization of a sample may also be used and are described further belowand for example in U.S. Pat. No. 9,184,036, the content of which isincorporated by reference herein in its entirety.

The essentially non-absorbent membrane may be associated with a housing,which may be the microwell, in which the essentially non-absorbentmembrane may be positioned, for example, at a top or a bottom of thehousing. The housing may be constructed of any suitable material that iscompatible with the material of the essentially non-absorbent membrane.Examples of such materials include, by way of example and notlimitation, any of the materials listed above for the essentiallynon-absorbent membrane. The material for the housing and for theessentially non-absorbent membrane may be the same or may be different.In some examples, the essentially non-absorbent membrane is part of amicrowell.

As mentioned above, in some examples the essentially non-absorbentmembrane is part of a microwell or an array of microwells. Theessentially non-absorbent membranes of at least two of the microwellsmay comprise liquid samples, which may be the same or different, and theelectrical field may be activated to selectively release droplets fromeach of the essentially non-absorbent membranes of the at least twomicrowells. The top or the bottom of the microwell may comprise theessentially non-absorbent membrane. The volume of the microwell isdependent on the nature of the liquid samples, the nature of the pore,the nature and size of the essentially non-absorbent membrane, the spraysolvent, the capture particle or cell, the analyte concentrations, themass label concentration, for example. In some examples the volume ofthe microwell is about 1 femtoliter(s) (fL) to about 100 microliters(μL), or about 1 μL to about 100 nanoliters (nL), or about 1 μL to about50 nL, or about 1 μL to about 10 nL, or about 1 μL to about 5 nL, orabout 1 μL to about 1 nL, or about 1 nL to about 2 nL. In some examples,where the microwells are circular, the diameter of the microwell isabout 5 micrometers (pm) to about 40 millimeters (mm), or about 5 μm toabout 500 μm, or about 500 μm to about 2 mm, or about 2 mm to about 40mm. The microwell around a single pore can hold a defined volume ofliquid, which allows a defined spray liquid volume and therefore a fixedhigh concentration of an analyte and short desorption time of the liquidwithin the pore.

The array can comprise 2 to about 100,000 microwells, or 2 to about50,000 microwells, or 2 to about 10,000 microwells, or 2 to about 5,000microwells, or 2 to about 2,500 microwells, or 2 to about 1,000microwells, or 2 to about 500 microwells, or 2 to about 100 microwells,or 2 to about 50 microwells, or about 10 to about 100,000 microwells, orabout 10 to about 50,000 microwells, or about 10 to about 10,000microwells, or about 10 to about 5,000 microwells, or about 10 to about2,500 microwells, or about 10 to about 1,000 microwells, or about 100 toabout 10,000 microwells, or about 100 to about 5,000 microwells, orabout 100 to about 2,500 microwells, or about 5,000 to about 10,000microwells, or about 2,500 to about 7,500 microwells, for example.

An array of apparatus 10 in an example in accordance with the principlesdescribed herein is depicted in FIG. 5 . Array 70 is shown comprising24×32 grid (768) of apparatus 10, each comprising a microwell 14.

As mentioned above, an array of microwells and an electric fieldgenerator may be disposed to one another such that one or both may bemoved in such a manner as to selectively activate an electric field forone or more of the microwells. An example, by way of illustration andnot limitation, is shown in FIGS. 6-7 . Apparatus 80 comprises array 70and electric field generator 74. Robotic arm 72 controls the movement ofarray 70 and, optionally, robotic arm 76 controls the movement ofelectric field generator 74. Apparatus 80 also comprises a housing (notshown), which provides support for one or both of robotic arms 72 and76. Each of robotic arm 72 and robotic arm 76 are separatelycontrollable using suitable electronics and controllers (not shown) suchthat one or both of array 70 and electric field generator 74 may bemoved with respect to one another. The mass spectrometer inlet isaligned with movement of the electric field generator. In that manner anelectric field may be applied selectively to one or more of apparatus 80comprising array 70 thereby allowing for interrogation of specificregion(s) of the essentially non-absorbent membrane of the microwells ofarray 70. Ionization of droplets may be achieved from distinct regionsof the essentially non-absorbent membrane by application of anelectrical potential to that region only or by using externalstructures, including nanostructures, to facilitate ionization fromselected regions. Furthermore, array 70 may be disposed with respect tothe intake of a mass spectrometer so that droplets of liquid selectivelyreleased from the membranes may be subjected to mass spectral analysis(see FIG. 4 ).

It should be noted that in the example shown in FIGS. 6-7 , the roboticarm controlling electric field generator 74 is shown above array 70.This is by way of illustration only; in some examples robotic arm 76 maybe below array 70 or adjacent (on the side) of array 70, for example.

The apparatuses of the invention have application in any situation inwhich release of precise small volumes of liquid on a membrane isdesired. Examples of such applications include, by way of illustrationand not limitation, detection of target rare molecules, non-raremolecules, non-rare cells and rare cells, for example. In some examples,the essentially non-absorbent membrane comprises more than one pore andthe electrical field is activated to selectively release droplets froman individual pore or subset of pores. The released droplets aresubjected to mass spectrometry analysis to determine an area adjacentthe individual pore or subset of pores where a particular MS label islocated. The liquid on the membrane corresponding to the area is removedfor analysis by methods discussed more fully below. The liquid adjacentthe individual pore may be removed by suction, punching out the area ofthe membrane, lifting, dissection, or extraction, for example, or acombination of two or more thereof.

Inductive Charging

In inductive electrospray ionization, as described for example in U.S.Pat. No. 9,184,036, the content of which is incorporated by referenceherein in its entirety, a potential may be applied to one or moreelectrodes (e.g., the electric field generator) placed close to theessentially non-absorbent membrane that contains the sample. It pulsesrepeatedly in either the positive or negative mode at a frequencyranging from 10-2000 Hz. Strong dynamic electromagnetic fields areproduced in the essentially non-absorbent membrane, and give the focusof the field, in the specifically targeted pore of the essentiallynon-absorbent membrane, resulting in a burst of charged droplets fromthe pore.

In inductive charging, the high voltage source (e.g., the electric fieldgenerator) is not in contact with sample or the essentiallynon-absorbent membrane that contains the sample. In this manner, theions are generated by inductive charging, i.e., an inductive method isused to charge the primary microdroplets. This allows for controlled andfocused droplet creation. The generated droplets are directed into themass spectrometer.

Charged droplet creation from a specific location on the essentiallynon-absorbent membrane can be achieved by placing an electrode (e.g.,the electric field generator) near the desired pore of the essentiallynon-absorbent membrane (typically 2-5 mm distant) and pulsing itrepetitively to high positive potentials (5-7 kV, 50-3,000 Hz, pulsewidth ˜0.2-2 ms). Electromagnetic induction produces high electricalfields in proximity to the specific pore of the essentiallynon-absorbent membrane that result in bursts of charged droplets fromonly that pore of the essentially non-absorbent membrane.

In some examples, liquid containing an MS label as discussed herein canbe directly discharged from an essentially non-absorbent membranebearing mass tagged rare cells or particles after applying a mass tagrelease agent. Accordingly, ambient electrostatic focusing of emittedcharged microdroplets/solvated ions to a smaller area such as theentrance to a mass spectrometer is achieved. In some examples,electrical field assisted charged droplet emission is achieved withnanofeatures provided by an array of points above or below theessentially non-absorbent membrane to provide a high electric fieldadjacent to a surface of the essentially non-absorbent membrane.

In some examples, intrinsic nanofeatures of the essentiallynon-absorbent membrane may be used to create a spray of analyte-bearingions from the wetted essentially non-absorbent membrane by chargeddroplet field emission. A combination of pneumatic and electrostaticforces may be employed to collect ions for subsequent analysis by a massspectrometer. This includes cases in which pneumatic forces are providedeither by suction from a mass spectrometer inlet (such as by vacuum) orby gas flow provided independent of a mass spectrometer.

Desorption Electrospray Ionization

One embodiment for generating an ion beam to be directed at the sampleon the essentially non-absorbent membrane employs Desorptionelectrospray ionization (DESI), which is described for example in Takatset al. (U.S. Pat. No. 7,335,897), the content of which is incorporatedby reference herein in its entirety. DESI allows ionizing and desorbinga material (analyte) at atmospheric or reduced pressure under ambientconditions. A DESI system generally includes a device for generating aDESI-active spray by delivering droplets of a liquid into a nebulizinggas. The system also includes a means for directing the DESI-activespray onto a surface. It is understood that the DESI-active spray may,at the point of contact with the surface, include both or either chargedand uncharged liquid droplets, gaseous ions, molecules of the nebulizinggas and of the atmosphere in the vicinity. The pneumatically assistedspray is directed onto the essentially non-absorbent membrane holdingthe sample where it interacts with one or more analytes, if present inthe sample, and generates desorbed ions of the analyte or analytes thatare ejected through the pore of the essentially non-absorbent membrane.The desorbed ions can be directed to a mass analyzer for mass analysis,to an IMS device for separation by size and measurement of resultingvoltage variations, to a flame spectrometer for spectral analysis, orthe like.

FIG. 11 illustrates schematically one embodiment of a DESI system 100.In this system, a spray 110 is generated by a conventional electrospraydevice 120. The device 120 includes a spray capillary 130 through whichthe liquid solvent 140 is fed. A surrounding nebulizer capillary 150forms an annular space through which a nebulizing gas such as nitrogen(N₂) is fed at high velocity. In one example, the liquid was awater/methanol mixture and the gas was nitrogen. A high voltage isapplied to the liquid solvent by a power supply 170 via a metalconnecting element. The result of the fast flowing nebulizing gasinteracting with the liquid leaving the capillary 130 is to form theDESI-active spray 110 comprising liquid droplets. DESI-active spray 110also may include neutral atmospheric molecules, nebulizing gas, andgaseous ions. Although an electrospray device 120 has been described,any device capable of generating a stream of liquid droplets carried bya nebulizing gas jet may be used to form the DESI-active spray 11.

The spray 110 is directed onto the essentially non-absorbent membraneholding the sample. The desorbed ions leaving the sample through thepore of the essentially non-absorbent membrane are collected andintroduced into the atmospheric inlet or interface of a massspectrometer for analysis. The essentially non-absorbent membrane may bea moveable platform or may be mounted on a moveable platform that can bemoved in the x, y or z directions by well-known drive means to desorband ionize the sample at different areas. Electric potential andtemperature of the platform may also be controlled by known means. Anyatmospheric interface that is normally found in mass spectrometers willbe suitable for use in the invention. Good results have been obtainedusing a typical heated capillary atmospheric interface. Good resultsalso have been obtained using an atmospheric interface that samples viaan extended flexible ion transfer line made either of metal or aninsulator.

Low Temperature Plasma

One embodiment for generating an ion beam to be directed at the sampleon the essentially non-absorbent membrane employs a low temperatureplasma (LTP) probe, which is described in Ouyang et al. (U.S. Pat. No.8,519,354), the content of each of which is incorporated by referenceherein in its entirety. Unlike electrospray or laser based ambientionization sources, plasma sources do not require an electrospraysolvent, auxiliary gases, and lasers. LTP can be characterized as anon-equilibrium plasma having high energy electrons, with relatively lowkinetic energy but reactive ions and neutrals; the result is a lowtemperature ambient plasma that can be used to desorb and ionizeanalytes from surfaces and produce molecular ions or fragment ions ofthe analytes. A distinguishing characteristic of the LTP, in comparisonwith high temperature (equilibrium) plasmas, is that the LTP does notbreakdown the molecules into atoms or small molecular fragments, so themolecular information is retained in the ions produced. LTP ionizationsources have the potential to be small in size, consume low power andgas (or to use only ambient air) and these advantages can lead toreduced operating costs. In addition to cost savings, LTP basedionization methods have the potential to be utilized with portable massspectrometers for real-time analytical analysis in the field (Gao, L.;Song, Q.; Patterson, G. E.; Cooks, D. Ouyang, Z., Anal. Chem. 2006, 78,5994-6002; Mulligan, C. C.; Talaty, N.; Cooks, R. G., ChemicalCommunications 2006, 1709-1711; and Mulligan, C. C.; Justes, D. R.;Noll, R. J.; Sanders, N. L.; Laughlin, B. C.; Cooks, R. G., The Analyst2006, 131, 556-567).

An exemplary LTP probe is shown in FIG. 12 . Such a probe may include ahousing having a discharge gas inlet port, a probe tip, two electrodes,and a dielectric barrier, in which the two electrodes are separated bythe dielectric barrier, and in which application of voltage from a powersupply generates an electric field and a low temperature plasma, inwhich the electric field, or gas flow, or both, propel the lowtemperature plasma out of the probe tip. The ionization source of theprobe described herein is based upon a dielectric barrier discharge(DBD; Kogelschatz, U., Plasma Chemistry and Plasma Processing 2003, 23,1-46). Dielectric barrier discharge is achieved by applying a highvoltage signal, for example an alternating current, between twoelectrodes separated by a dielectric barrier. A non-thermal, low power,plasma is created between the two electrodes, with the dielectriclimiting the displacement current. This plasma contains reactive ions,electrons, radicals, excited neutrals, and metastable species in theambient environment of the sample which can be used to desorb/ionizemolecules from a solid sample surface as well as ionizing liquids andgases. The plasma can be extracted from the discharge region anddirected toward the sample surface with the force by electric field, orthe combined force of the electric field and gas flow.

In certain embodiments, the probe further includes a power supply. Thepower supply can provide direct current or alternating current. Incertain embodiments, the power supply provides an alternating current.In certain embodiments, a discharge gas is supplied to the probe throughthe discharge gas inlet port, and the electric field and/or thedischarge gas propel the low temperature plasma out of the probe tip.The discharge gas can be any gas. Exemplary discharge gases includehelium, compressed or ambient air, nitrogen, and argon. In certainembodiments, the dielectric barrier is composed of an electricallyinsulating material. Exemplary electrically insulating materials includeglass, quartz, ceramics and polymers. In other embodiments, thedielectric barrier is a glass tube that is open at each end. In otherembodiments, varying the electric field adjusts the energy andfragmentation degree of ions generated from the analytes in a sample.

The plasma discharge from the low temperature plasma probe is directedonto the essentially non-absorbent membrane holding the sample. Theplasma interacts with the sample and causes a liquid droplet of thesample to be ejected through the pore of the essentially non-absorbentmembrane and introduced into the atmospheric inlet or interface of amass spectrometer for analysis.

Ionization Using wetted Porous Material

One embodiment for generating an ion beam to be directed at the sampleon the essentially non-absorbent membrane employs a probe comprised ofporous material that is wetted to produce ions, which is described inOuyang et al. (U.S. Pat. No. 8,859,956), the content of each of which isincorporated by reference herein in its entirety. An exemplary probe isshown in FIG. 13 . Porous materials, such as paper (e.g. filter paper orchromatographic paper) or other similar materials are used to hold andtransfer liquids an ion beam generated directly from the edges of thematerial when a high electric voltage is applied to the material. Theporous material is kept discrete (i.e., separate or disconnected) from aflow of solvent, such as a continuous flow of solvent. Instead, liquidis spotted onto the porous material. The spotted liquid is thenconnected to a high voltage source to produce an ion beam of the liquidthat is directed onto the essentially non-absorbent membrane holding thesample. The desorbed ions leaving the sample through the pore of theessentially non-absorbent membrane are collected and introduced into theatmospheric inlet or interface of a mass spectrometer for analysis. Theliquid is transported through the porous material without the need of aseparate solvent flow. Pneumatic assistance is not required; rather, avoltage is simply applied to the porous material.

In certain embodiments, the porous material is any cellulose-basedmaterial. In other embodiments, the porous material is a non-metallicporous material, such as cotton, linen wool, synthetic textiles, orplant tissue. In still other embodiments, the porous material is paper.Advantages of paper include: cost (paper is inexpensive); it is fullycommercialized and its physical and chemical properties can be adjusted;it can filter particulates (cells and dusts) from liquid samples; it iseasily shaped (e.g., easy to cut, tear, or fold); liquids flow in itunder capillary action (e.g., without external pumping and/or a powersupply); and it is disposable.

In certain embodiments, the porous material is integrated with a solidtip having a macroscopic angle that is optimized for spray.

In particular embodiments, the porous material is filter paper.Exemplary filter papers include cellulose filter paper, ashless filterpaper, nitrocellulose paper, glass microfiber filter paper, andpolyethylene paper. Filter paper having any pore size may be used.Exemplary pore sizes include Grade 1 (11 μm), Grade 2 (8 μm), Grade 595(4-7 μm), and Grade 6 (3 μm). Pore size will not only influence thetransport of liquid inside the spray materials, but could also affectthe formation of the Taylor cone at the tip. The optimum pore size willgenerate a stable Taylor cone and reduce liquid evaporation. The poresize of the filter paper is also an important parameter in filtration,i.e., the paper acts as an online pretreatment device. Commerciallyavailable ultra filtration membranes of regenerated cellulose, with poresizes in the low nm range, are designed to retain particles as small as1000 Da. Ultra filtration membranes can be commercially obtained withmolecular weight cutoffs ranging from 1000 Da to 100,000 Da.

Probes of the invention work well for the generation of micron scaledroplets simply based on using the high electric field generated at anedge of the porous material. In particular embodiments, the porousmaterial is shaped to have a macroscopically sharp point, such as apoint of a triangle, for ion generation. Probes of the invention mayhave different tip widths. In certain embodiments, the probe tip widthis at least about 5 μm or wider, at least about 10 μm or wider, at leastabout 50 μm or wider, at least about 150 μm or wider, at least about 250μm or wider, at least about 350 μm or wider, at least about 400μ orwider, at least about 450 μm or wider, etc. In particular embodiments,the tip width is at least 350 μm or wider. In other embodiments, theprobe tip width is about 400 μm. In other embodiments, probes of theinvention have a three dimensional shape, such as a conical shape.

Detection of Target Rare Molecules

In some examples, the apparatuses of the invention are used in thedetection of different populations of target rare molecules employingaffinity agents and different labels that are detectable using MStechniques. In some examples, one or more alteration agents are used togenerate MS labels that are chosen to differentiate among differentpopulations of target rare molecules. The methods also employ separationmethods, in which liquid droplets are produced and are examined by MStechniques for one or both of the presence and amount of each differentMS label. Differentiation of the MS labels yields information about oneor both of the presence and amount of each different population oftarget rare molecules. The number of MS labels may be as many as 10⁶ ormore per target rare molecule or as few as 10 per target rare molecule.The number of MS labels per target rare molecule may be about 10 toabout 10¹², or about 10 to about 10¹⁰, or about 10 to about 10⁸, orabout 10 to about 10⁶, or about 10 to about 10⁴, or about 10 to about100, or about 100 to about 10¹⁰, or about 100 to about 10⁸, or about 100to about 10⁶, or about 100 to about 10⁴, for example.

In some examples, the methods are for detecting one or more differentpopulations of target rare molecules in a sample suspected of containingthe one or more different populations of rare molecules and non-raremolecules. The sample in liquid form is contacted to a microwell thatcomprises an essentially non-absorbent membrane. Optionally, theconcentration of the one or more different populations of target raremolecules is enhanced over that of the non-rare molecules to form aconcentrated sample by employing a suitable technique such as, forexample, filtration. The sample is incubated with, for each differentpopulation of target rare molecules, an affinity agent that comprises aspecific binding partner that is specific for and binds to a target raremolecule of one of the populations of the target rare molecules. Theaffinity agent comprises a mass spectrometry label precursor or a firstalteration agent. The affinity agent may be non-particulate orparticulate. The first alteration agent either facilitates the formationof a mass spectrometry label from the mass spectrometry label precursoror releases an entity that comprises the mass spectrometry labelprecursor from the affinity agent. If the first alteration agent doesnot facilitate the formation of a mass spectrometry label from the massspectrometry label precursor, the sample is subjected to a secondalteration agent that facilitates the formation of a mass spectrometrylabel from the mass spectrometry label precursor. The mass spectrometrylabel corresponds to or comprises one of the populations of target raremolecules. The sample on the essentially non-absorbent membrane isexposed to an electrical field to release droplets of the sample throughthe at least one pore of the essentially non-absorbent membrane. Thedroplets are subjected to mass spectrometry analysis to determine thepresence and/or amount of each different mass spectrometry label. Thepresence and/or amount of each different mass spectrometry label to thepresent and/or amount of each different population of target raremolecules in the sample for each microwell.

In one approach, particle amplification is utilized and provides foraggregating or clustering particles to form particle aggregates. In oneexample, a larger particle (carrier particle) can be coated by manysmaller particles (label particles). To further achieve amplification,the carrier particle can be chained with other carrier particles usingone or more linking groups. The label particle contains the MS label onthe surface, which may be on the order of 10⁵ since the size of masslabel is comparatively small. In this approach, very low backgroundlevels are realized. The carrier particles and label particles shouldhave a diameter that is smaller than the pores in the essentiallynon-absorbent membrane.

It should be noted that one or more of the identification techniquesdiscussed below may be applied to a sample subsequent to contacting thesample with an essentially non-absorbent membrane in accordance with theprinciples described herein. Thus, approaches for analysis of samples toidentify one or more target rare molecules include first identifyingwhich microwells have target rare molecules of interest. Thus,techniques may be employed as a screening technique to identifymicrowells that have sample with target rare molecules for subsequentanalysis.

The sample to be analyzed is one that is suspected of containing targetrare molecules, non-rare cells and rare cells. The samples may bebiological samples or non-biological samples. Biological samples may befrom a mammalian subject or a non-mammalian subject. Mammalian subjectsmay be, e.g., humans or other animal species. Biological samples includebiological fluids such as whole blood, serum, plasma, sputum, lymphaticfluid, semen, vaginal mucus, feces, urine, spinal fluid, saliva, stool,cerebral spinal fluid, tears, and mucus, for example. Biological tissueincludes, by way of illustration, hair, skin, sections or excisedtissues from organs or other body parts, for example. In many instances,the sample is whole blood, plasma or serum. Rare cells may be from, forexample, lung, bronchus, colon, rectum, pancreas, prostate, breast,liver, bile duct, bladder, ovary, brain, central nervous system, kidney,pelvis, uterine corpus, oral cavity or pharynx or melanoma cancers. Therare cells may be, but are not limited to, pathogens such as bacteria,virus, fungus, and protozoa; malignant cells such as malignant neoplasmsor cancer cells; circulating endothelial cells; circulating tumor cells;circulating cancer stem cells; circulating cancer mesochymal cells;circulating epithelial cells; fetal cells; immune cells (B cells, Tcells, macrophages, NK cells, monocytes); and stem cells; for example.In some examples, the sample to be tested is a blood sample from amammal such as, but not limited to, a human subject. The blood sample isone that contains cells such as, for example, non-rare cells and rarecells. In some examples the blood sample is whole blood or plasma.

The phrase “target rare molecule” refers to a molecule includingbiomarkers that may be detected in a sample where the molecule orbiomarker is indicative of a particular population of cells. Target raremolecules include, but are not limited to, antigens (such as, forexample, proteins, peptides, hormones, vitamins, allergens, autoimmuneantigens, carbohydrates, lipids, glycoproteins, co-factors, antibodies,and enzymes) and nucleic acids.

The phrase “population of target rare molecules” refers to a group ofmolecules that share a common antigen or nucleic acid that is specificfor the group of molecules. The phrase “specific for” means that thecommon antigen or nucleic acid distinguishes the group of molecules fromother molecules.

Non-rare molecules are present in relatively large amounts when comparedto an amount of rare molecules in a sample.

The phrase “population of cells” refers to a group of cells having anantigen or nucleic acid on their surface or inside the cell in which theantigen is common to all of the cells of the group and where the antigenis specific for the group of cells.

Rare cells are those cells that are present in a sample in relativelysmall quantities when compared to the amount of non-rare cells in asample. In some examples, the rare cells are present in an amount ofabout 10⁻⁸% to about 10⁻²% by weight of a total cell population in asample suspected of containing the rare cells. The rare cells may be,but are not limited to, malignant cells such as malignant neoplasms orcancer cells; circulating endothelial cells; circulating epithelialcells; mesochymal cells; fetal cells; immune cells (B cells, T cells,macrophages, NK cells, monocytes); stem cells; nucleated red blood cells(normoblasts or erythroblasts); and immature granulocytes.

Non-rare cells are those cells that are present in relatively largeamounts when compared to the amount of rare cells in a sample. In someexamples, the non-rare cells are at least about 10 times, or at leastabout 10² times, or at least about 10³ times, or at least about 10⁴times, or at least about 10⁵ times, or at least about 10⁶ times, or atleast about 10⁷ times, or at least about 10⁸ times greater than theamount of the rare cells in the total cell population in a samplesuspected of containing non-rare cells and rare cells. The non-rarecells may be, but are not limited to, white blood cells, platelets, andred blood cells, for example.

Target rare molecules of rare cells include, but are not limited to,cancer cell type biomarkers, oncoproteins and oncogenes, chemoresistance biomarkers, metastatic potential biomarkers, and cell typingmarkers, for example. Cancer cell type biomarkers include, by way ofillustration and not limitation, cytokeratins (CK) (CK1, CK2, CK3, CK4,CKS, CK6, CK7, CK8 and CK9, CK10, CK12, CK 13, CK14, CK16, CK17, CK18,CK19 and CK2), epithelial cell adhesion molecule (EpCAM), N-cadherin,E-cadherin and vimentin, for example. Oncoproteins and oncogenes withlikely therapeutic relevance due to mutations include, but are notlimited to, WAF, BAX-1, PDGF, JAGGED 1, NOTCH, VEGF, VEGHR, CA1X, MIB1,MDM, PR, ER, SELS, SEMI, PI3K, AKT2, TWIST1, EML-4, DRAFF, C-MET, ABL1,EGFR, GNAS, MLH1, RET, MEK1, AKT1, ERBB2, HER2, HNF1A, MPL, SMAD4, ALK,ERBB4, HRAS, NOTCH1, SMARCB1, APC, FBXW7, IDH1, NPM1, SMO, ATM, FGFR1,JAK2, NRAS, SRC, BRAF, FGFR2, JAK3, RA, STK11, CDH1, FGFR3, KDR, PIK3CA,TP53, CDKN2A, FLT3, KIT, PTEN, VHL, CSF1R, GNA11, KRAS, PTPN11, DDR2,CTNNB1, GNAQ, MET, RB1, AKT1, BRAF, DDR2, MEK1, NRAS, FGFR1, and ROS1,for example.

Endothelial cell typing markers include, by way of illustration and notlimitation, CD136, CD105/Endoglin, CD144/VE-cadherin, CD145, CD34, Cd41CD136, CD34, CD90, CD31/PECAM-1, ESAM,VEGFR2/Fik-1, Tie-2, CD202b/TEK,CD56/NCAM, CD73/VAP-2, claudin 5, Z0-1, and vimentin, for example.

Metastatic potential biomarkers include, but are limited to, urokinaseplasminogen activator (uPA), plasminogen activator inhibitor (PAI-1),CD95, serine proteases (e.g., plasmin and ADAM, for example); serineprotease inhibitors (e.g., Bikunin); matrix metalloproteinases (e.g.,MMP9); matrix metalloproteinase inhibitors (e.g., TIMP-1).Chemoresistance biomarkers include, by way of illustration and notlimitation, PL2L piwi like, 5T4, ADLH, β-integrin, α6 integrin, c-kit,c-met, LIF-R, CXCR4, ESA, CD 20, CD44, CD133, CKS, TRAF2 and ABCtransporters, cancer cells that lack CD45 or CD31 but contain CD34 areindicative of a cancer stem cell; and cancer cells that contain CD44 butlack CD24.

In methods herein, white blood cells may be excluded as non-rare cells.For example, markers such as, but not limited to, CD45, CTLA-4, CD4,CD6S and CDS that are present on white blood cells can be used toindicate that a cell is not a rare cell of interest. In a particularnon-limiting example, CD45 antigen (also known as protein tyrosinephosphatase receptor type C or PTPRC) and originally called leukocytecommon antigen is useful in detecting all white blood cells.

Additionally, CD45 can be used to differentiate different types of whiteblood cells that might be considered rare cells. For example,granulocytes are indicated by CD45+, CD15+; monocytes are indicated byCD45+, CD14+; T lymphocytes are indicated by CD45+, CD3+; T helper cellsare indicated by CD45+,CD3+, CD4+; cytotoxic T cells are indicated byCD45+,CD3+, CDS+; β-lymphocytes are indicated by CD45+, CD19+ or CD45+,CD20+; thrombocytes are indicated by CD45+, CD61+; and natural killercells are indicated by CD16+, CD56+, and CD3−. Furthermore, two commonlyused CD molecules, namely, CD4 and CD8, are, in general, used as markersfor helper and cytotoxic T cells, respectively. These molecules aredefined in combination with CD3+, as some other leukocytes also expressthese CD molecules (some macrophages express low levels of CD4;dendritic cells express high levels of CDS).

In other cases the rare cell is a pathogen, which includes, but is notlimited to, gram-positive bacteria (e.g., Enterococcus sp. Group Bstreptococcus, Coagulase-negative staphylococcus sp. Streptococcusviridans, Staphylococcus aureus and saprophyicus, Lactobacillus andresistant strains thereof, for example); yeasts including, but notlimited to, Candida albicans, for example; gram-negative bacteria suchas, but not limited to, Escherichia coli, Klebsiella pneumoniae,Citrobacter koseri, Citrobacter freundii, Klebsiella oxytoca, Morganellamorganii, Pseudomonas aeruginosa, Proteus mirabilis, Serratiamarcescens, and Diphtheroids (gnb) and resistant strains thereof, forexample; viruses such as, but not limited to, HIV, HPV, Flu, and MERSA,for example; and sexually transmitted diseases. In the case of detectingrare cell pathogens, a particle reagent is added that comprises abinding partner, which binds to the rare cell pathogen population.Additionally, for each population of cellular target rare molecules onthe pathogen, a reagent is added that comprises a binding partner forthe cellular target rare molecule, which binds to the cellular targetrare molecules in the population.

The phrase “non-cellular target rare molecules” refers to target raremolecules that are not bound to a cell and/or that freely circulate in asample. Such non-cellular target rare molecules include biomoleculesuseful in medical diagnosis of diseases, which include, but are notlimited to, biomarkers for detection of cancer, cardiac damage,cardiovascular disease, neurological disease, hemostasis/hemastasis,fetal maternal assessment, fertility, bone status, hormone levels,vitamins, allergies, autoimmune diseases, hypertension, kidney disease,diabetes, liver diseases, infectious diseases and other biomoleculesuseful in medical diagnosis of diseases, for example.

As mentioned above, in some instances, one or more of the populations oftarget rare molecules may be a population of non-cellular target raremolecules. In such an instance, for each population of non-cellulartarget rare molecules, a capture particle entity is added that comprisesa binding partner for the non-cellular target rare molecule, which bindsto the non-cellular target rare molecules in the population to formparticle-bound non-cellular target rare molecules thereby rendering anon-cellular target rare molecule in particulate form for purposes ofcarrying out an enhancement of a concentration of one or differentpopulations of a non-cellular target rare molecule over that of non-raremolecules to form a concentrated sample.

The composition of the particle may be organic or inorganic, magnetic ornon-magnetic. Organic polymers include, by way of illustration and notlimitation, nitrocellulose, cellulose acetate, poly(vinyl chloride),polyacrylamide, polyacrylate, polyethylene, polypropylene,poly(4-methylbutene), polystyrene, poly(methyl methacrylate),poly(hydroxyethyl methacrylate),poly(styrene/divinylbenzene),poly(styrene/acrylate), poly(ethyleneterephthalate), melamine resin, nylon, poly(vinyl butyrate), forexample, either used by themselves or in conjunction with othermaterials and including latex, microparticle and nanoparticle formsthereof. The particles may also comprise carbon (e.g., carbonnanotubes), metal (e.g., gold, silver, and iron, including metal oxidesthereof), colloids, dendrimers, dendrons, nucleic acids, Branchchain-DNA, and liposomes, for example.

The diameter of the particles of the particle entity is dependent on oneor more of the nature of the target rare molecule, the nature of thesample, the nature and the pore size of the essentially non-absorbentmembrane, the adhesion of the particle to the membrane, the surface ofthe particle, the surface of the essentially non-absorbent membrane, theliquid ionic strength, liquid surface tension and components in theliquid, and the number, size, shape and molecular structure of attachedaffinity agent and MS label precursors, for example. The diameter of theparticles must be large enough to reduce background contribution to anacceptable level but not so large as to achieve inefficient separationof the particles from non-rare molecules. In some examples in accordancewith the principles described herein, the average diameter of theparticles should be at least about 0.02 microns (20 nm) and not morethan about 200 microns, or not more than about 120 microns. In someexamples, the particles have an average diameter from about 0.1 micronsto about 20 microns, or about 0.1 microns to about 15 microns, or about0.1 microns to about 10 microns, or about 0.02 microns to about 0.2microns, or about 0.2 microns to about 1 micron, or about 1 micron toabout 5 microns, or about 1 micron to about 20 microns, or about 1micron to about 15 microns, or about 1 micron to about 10 microns, orabout 5 microns to about 20 microns, or about 5 to about 15 microns, orabout 5 to about 10 microns, or about 6 to about 15 microns, or about 6to about 10 microns, for example. In some examples, the adhesion of theparticles to the surface is so strong that the particle diameter can besmaller than the pore size of the essentially non-absorbent membrane. Inother examples, the particles are sufficiently larger than the pore sizeof the essentially non-absorbent membrane such that physically theparticles cannot fall through the pores of the essentially non-absorbentmembrane.

The capture particle entity also includes a binding partner that isspecific for the non-cellular target rare molecule. The phrase “bindingpartner” refers to a molecule that is a member of a specific bindingpair. A member of a specific binding pair is one of two differentmolecules having an area on the surface or in a cavity, whichspecifically binds to and is thereby defined as complementary with aparticular spatial and polar organization of the other molecule. Themembers of the specific binding pair may be members of an immunologicalpair such as antigen-antibody or hapten-antibody, biotin-avidin,hormones-hormone receptors, enzyme-substrate, nucleic acid duplexes,IgG-protein A, and polynucleotide pairs such as DNA-DNA or DNA-RNA. Thebinding partner may be bound, either covalently or non-covalently, tothe particle of the particle reagent. “Non-covalently” means that thebinding partner is bound to the particle as the result of one or more ofhydrogen bonding, van der Waals forces, electrostatic forces,hydrophobic effects, physical entrapment in the particles, and chargedinteractions. “Covalently” means that the binding partner is bound tothe particle by a bond or a linking group, which may be aliphatic oraromatic and may comprise a chain of 2 to about 60 or more atoms thatinclude carbon, oxygen, sulfur, nitrogen and phosphorus.

In some examples, samples are collected from a body of a subject into asuitable container such as, but not limited to, a cup, a bag, a bottle,capillary, or a needle, for example. Blood samples may be collected intoa VACUTAINER (blood collection tube, commercially available from BD).The container may contain a collection medium into which the sample isdelivered. The collection medium is usually a dry medium and maycomprise an amount of platelet deactivation agent effective to achievedeactivation of platelets in the blood sample when mixed with the bloodsample.

Platelet deactivation agents include, but are not limited to, chelatingagents such as, agents that comprise a triacetic acid moiety or a saltthereof, a tetraacetic acid moiety or a salt thereof, a pentaacetic acidmoiety or a salt thereof, or a hexaacetic acid moiety or a salt thereof.In some examples, the chelating agent is ethylene diamine tetraaceticacid (EDTA) and its salts or ethylene glycol tetraacetate (EGTA) and itssalts. The effective amount of platelet deactivation agent is dependenton one or more of the nature of the platelet deactivation agent, thenature of the blood sample, level of platelet activation and ionicstrength, for example. In some examples, for EDTA as the anti-plateletagent, the amount of dry EDTA in the container is that which willproduce a concentration of about 1.0 to about 2.0 mg/mL of blood, orabout 1.5 mg/mL of the blood. The amount of the platelet deactivationagent is that which is sufficient to achieve at least about 90%, or atleast about 95%, or at least about 99% of platelet deactivation.

As mentioned above, optionally, the concentration of the one or moredifferent populations of target rare molecules is enhanced over that ofthe non-rare molecules to form a concentrated sample. In some examples,prior to contacting the sample with an essentially non-absorbentmembrane, the sample is subjected to a filtration procedure using aporous matrix that retains the target rare molecules while allowing thenon-rare molecules to pass through the porous matrix thereby enhancingthe concentration of the target rare molecules. In the event that one ormore target rare molecules are non-cellular, i.e., not associated with acell or other biological particle, the sample is combined with one ormore capture particle entities wherein each capture particle entitycomprises a binding partner for the non-cellular target rare molecule ofeach of the populations of non-cellular target rare molecules to renderthe non-cellular target rare molecules in particulate form, i.e., toform particle-bound non-cellular target rare molecules. The combinationof the sample and the capture particle entities is held for a period oftime and at a temperature to permit the binding of non-cellular targetrare molecules with corresponding binding partners of the captureparticle entities. Moderate temperatures are normally employed, whichmay range from about 5° C. to about 70° C. or from about 15° C. to about70° C. or from about 20° C. to about 45° C. The time period for anincubation period is about 0.2 seconds to about 6 hours, or about 2seconds to about 1 hour, or about 1 to about 5 minutes, for example.

The time period for contact of the sample to the essentiallynon-absorbent membrane may be dependent for example on one or more ofthe nature and size of the different populations of target rare cellsand/or particle-bound target rare molecules, the nature of theessentially non-absorbent membrane, the size of the pores of theessentially non-absorbent membrane, the level of vacuum applied to thesample on the essentially non-absorbent membrane, the volume to befiltered, and the surface area of the essentially non-absorbentmembrane. In some examples, the period of contact is about 1 minute toabout 1 hour, about 5 minutes to about 1 hour, or about 5 minutes toabout 45 minutes, or about 5 minutes to about 30 minutes, or about 5minutes to about 20 minutes, or about 5 minutes to about 10 minutes, orabout 10 minutes to about 1 hour, or about 10 minutes to about 45minutes, or about 10 minutes to about 30 minutes, or about 10 minutes toabout 20 minutes.

In methods herein, the sample, either unconcentrated or concentrated,may be incubated with, for each different population of target raremolecules, an affinity agent that comprises a binding partner that isspecific for and binds to a target rare molecule of one of thepopulations of the target rare molecules. The affinity agent alsocomprises an MS label precursor or a first alteration agent thatfacilitates the formation of an MS label from each different MS labelprecursor or that releases an entity that comprises the MS labelprecursor from the affinity agent. In many examples, the abovecombination is provided in an aqueous medium, which may be solely wateror which may also contain organic solvents such as, for example, polaraprotic solvents, polar protic solvents such as, e.g., dimethylsulfoxide(DMSO), dimethylformamide (DMF), acetonitrile, an organic acid, or analcohol, and non-polar solvents miscible with water such as, e.g.,dioxene, in an amount of about 0.1% to about 50%, or about 1% to about50%, or about 5% to about 50%, or about 1% to about 40%, or about 1% toabout 30%, or about 1% to about 20%, or about 1% to about 10%, or about5% to about 40%, or about 5% to about 30%, or about 5% to about 20%, orabout 5% to about 10%, by volume. In some examples, the pH for theaqueous medium is usually a moderate pH. In some examples, the pH of theaqueous medium is about 5 to about 8, or about 6 to about 8, or about 7to about 8, or about 5 to about 7, or about 6 to about 7, orphysiological pH. Various buffers may be used to achieve the desired pHand maintain the pH during any incubation period. Illustrative buffersinclude, but are not limited to, borate, phosphate (e.g., phosphatebuffered saline), carbonate, TRIS, barbital, PIPES, HEPES, MES, ACES,MOPS, and BICINE.

An amount of aqueous medium employed is dependent on a number of factorssuch as, but not limited to, the nature and amount of the sample, thenature and amount of the reagents, the stability of target rare cells,and the stability of target rare molecules. In some examples, the amountof aqueous medium per 10 mL of sample is about 5 mL to about 100 mL, orabout 5 mL to about 80 mL, or about 5 mL to about 60 mL, or about 5 mLto about 50 mL, or about 5 mL to about 30 mL, or about 5 mL to about 20mL, or about 5 mL to about 10 mL, or about 10 mL to about 100 mL, orabout 10 mL to about 80 mL, or about 10 mL to about 60 mL, or about 10mL to about 50 mL, or about 10 mL to about 30 mL, or about 10 mL toabout 20 mL, or about 20 mL to about 100 mL, or about 20 mL to about 80mL, or about 20 mL to about 60 mL, or about 20 mL to about 50 mL, orabout 20 mL to about 30 mL.

Where one or more of the target rare molecules are part of a cell, theaqueous medium may also comprise a lysing agent for lysing of cells. Alysing agent is a compound or mixture of compounds that disrupt theintegrity of the membranes of cells thereby releasing intracellularcontents of the cells. Examples of lysing agents include, but are notlimited to, non-ionic detergents, anionic detergents, amphotericdetergents, low ionic strength aqueous solutions (hypotonic solutions),bacterial agents, aliphatic aldehydes, and antibodies that causecomplement dependent lysis, for example. Various ancillary materials maybe present in the dilution medium. All of the materials in the aqueousmedium are present in a concentration or amount sufficient to achievethe desired effect or function.

In some examples, where one or more of the target rare molecules arepart of a cell, it may be desirable to fix the cells of the sample.Fixation of the cells immobilizes the cells and preserves cell structureand maintains the cells in a condition that closely resembles the cellsin an in vivo-like condition and one in which the antigens of interestare able to be recognized by a specific affinity agent. The amount offixative employed is that which preserves the cells but does not lead toerroneous results in a subsequent assay. The amount of fixative maydepend for example on one or more of the nature of the fixative and thenature of the cells. In some examples, the amount of fixative is about0.05% to about 0.15% or about 0.05% to about 0.10%, or about 0.10% toabout 0.15% by weight. Agents for carrying out fixation of the cellsinclude, but are not limited to, cross-linking agents such as, forexample, an aldehyde reagent (such as, e.g., formaldehyde,glutaraldehyde, and paraformaldehyde,); an alcohol (such as, e.g., C1-C5alcohols such as methanol, ethanol and isopropanol); a ketone (such as aC3-C5 ketone such as acetone); for example. The designations C1-C5 orC3-C5 refer to the number of carbon atoms in the alcohol or ketone. Oneor more washing steps may be carried out on the fixed cells using abuffered aqueous medium.

If necessary after fixation, the cell preparation may also be subjectedto permeabilization. In some instances, a fixation agent such as, analcohol (e.g., methanol or ethanol) or a ketone (e.g., acetone), alsoresults in permeabilization and no additional permeabilization step isnecessary. Permeabilization provides access through the cell membrane totarget molecules of interest. The amount of permeabilization agentemployed is that which disrupts the cell membrane and permits access tothe target molecules. The amount of permeabilization agent depends onone or more of the nature of the permeabilization agent and the natureand amount of the cells. In some examples, the amount ofpermeabilization agent is about 0.01% to about 10%, or about 0.1% toabout 10%. Agents for carrying out permeabilization of the cellsinclude, but are not limited to, an alcohol (such as, e.g., C1-C5alcohols such as methanol and ethanol); a ketone (such as a C3-C5 ketonesuch as acetone); a detergent (such as, e.g., saponin, TRITON X-100(4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol,t-Octylphenoxypolyethoxyethanol, Polyethylene glycol tert-octylphenylether buffer, commercially available from Sigma Aldrich), and TWEEN-20(Polysorbate 20, commercially available from Sigma Aldrich)). One ormore washing steps may be carried out on the permeabilized cells using abuffered aqueous medium.

As mentioned above, an affinity agent employed in methods herein is onethat is specific for a target rare molecule. The affinity agent is amember of a specific binding pair, which is one of two differentmolecules, having an area on the surface or in a cavity, whichspecifically binds to and is thereby defined as complementary with aparticular spatial and polar organization of the other molecule. Themembers of the specific binding pair may be members of an immunologicalpair such as antigen-antibody and hapten-antibody, although otherspecific binding pairs include, for example, biotin-avidin,hormones-hormone receptors, enzyme-substrate, aptamers, nucleic acidduplexes, IgG-protein A, and nucleic acid pairs such as DNA-DNA,DNA-RNA. In the case of cells, the affinity agent is an agent thatspecifically recognizes or binds to a target molecule antigen associatedwith a cell.

Specific binding involves the specific recognition of one of twodifferent molecules for the other compared to substantially lessrecognition of other molecules. On the other hand, non-specific bindinginvolves non-covalent binding between molecules that is relativelyindependent of specific surface structures. Non-specific binding mayresult from several factors including hydrophobic interactions betweenmolecules.

Antibodies specific for a target molecule for use in immunoassays toidentify cells can be monoclonal or polyclonal. Such antibodies can beprepared by techniques that are well known in the art such asimmunization of a host and collection of sera (polyclonal) or bypreparing continuous hybrid cell lines and collecting the secretedprotein (monoclonal) or by cloning and expressing nucleotide sequencesor mutagenized versions thereof coding at least for the amino acidsequences required for specific binding of natural antibodies.

Antibodies may include a complete immunoglobulin or fragment thereof,which immunoglobulins include the various classes and isotypes, such asIgA, IgD, IgE, IgG1, IgG2a, IgG2b and IgG3, IgM, etc. Fragments thereofmay include Fab, Fv and F(ab')₂, and Fab', for example. In addition,aggregates, polymers, and conjugates of immunoglobulins or theirfragments can be used where appropriate so long as binding affinity fora particular molecule is maintained.

Polyclonal antibodies and monoclonal antibodies may be prepared bytechniques that are well known in the art. For example, in one approachmonoclonal antibodies are obtained by somatic cell hybridizationtechniques. Monoclonal antibodies may be produced according to thestandard techniques of Köhler and Milstein, Nature 265:495-497, 1975.Reviews of monoclonal antibody techniques are found in LymphocyteHybridomas, ed. Melchers, et al. Springer-Verlag (New York 1978), Nature266: 495 (1977), Science 208: 692 (1980), and Methods of Enzymology 73(Part B): 3-46 (1981). In general, monoclonal antibodies can be purifiedby known techniques such as, but not limited to, chromatography, e.g.,DEAE chromatography, ABx chromatography, and HPLC chromatography; andfiltration, for example.

The affinity agent may be a nucleic acid (e.g., polynucleotide) that iscomplementary to a target nucleic acid. Polynucleotides refer to apolymeric form of nucleotides of any length, either deoxyribonucleotidesor ribonucleotides, or analogs thereof. The following are non-limitingexamples of polynucleotides: coding or non-coding regions of a gene orgene fragment, loci (locus) defined from linkage analysis, exons,introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes,cDNA, recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers. A polynucleotide may comprise modifiednucleotides such as, for example, methylated nucleotides and nucleotideanalogs. If present, modifications to the nucleotide structure may beimparted before or after assembly of the polymer. The sequence ofnucleotides may be interrupted by non-nucleotide components. Apolynucleotide may be further modified, such as by conjugation with alabeling component.

The affinity agent comprises either an MS label precursor or analteration agent that facilitates the formation of an MS label from anMS label precursor where the MS label corresponds to a target raremolecule of one of the populations of target rare molecules. The MSlabel allows differentiation of one of the populations of target raremolecules from other populations of rare molecules. Furthermore,selection of the MS label may be carried out to avoid overlapping massesin the analysis by MS, to avoid background interference in the MSanalysis, and to permit multiplexing.

The phrase “mass spectrometry label” or “MS label” refers to one or agroup of molecules having unique masses, preferably below 3 kDA, suchthat each unique mass, corresponds to, and is used to determine apresence and/or amount of, each different population of target raremolecules. The MS labels are molecules of defined mass and include, butare not limited to, polypeptides, polymers, fatty acids, carbohydrates,organic amines, nucleic acids, and organic alcohols, for example, whosemass can be varied by substitution and chain size, for example. In thecase of polymeric materials, the number repeating units is adjusted suchthat the mass is in a region that does not overlap with a backgroundmass from the sample. The phrase “MS label” also includes an analytethat is captured by an affinity particle, a derivatized analyte wherethe derivatization renders the analyte ionic, and an underivatizedanalyte in ionic form. The MS label generates a unique mass pattern dueto structure and fragmentation upon ionization.

The term “analyte” refers to a molecule or molecules that are todetected. Exemplary analytes by way of illustration and not limitation,include drugs, metabolites, pesticides and pollutants. Representativeanalytes, by way of illustration and not limitation, also includealkaloids, steroids, lactams, aminoalkylbenzenes, benzheterocyclics,purines, drugs derived from marijuana, hormones, polypeptides whichincludes proteins, immunosuppressants, vitamins, prostaglandins,tricyclic antidepressants, anti-neoplastics, nucleosides and nucleotidesincluding polynucleosides and polynucleotides, miscellaneous individualdrugs which include methadone, meprobamate, serotonin, meperidine,lidocaine, procainamide, acetylprocainamide, propranolol, griseofulvin,valproic acid, butyrophenones, antihistamines, chloramphenicol,anticholinergic drugs, and metabolites and derivatives of all of theabove. Also included are metabolites related to disease states,aminoglycosides, such as gentamicin, kanamicin, tobramycin, andamikacin, and pesticides such as, for example, polyhalogenatedbiphenyls, phosphate esters, thiophosphates, carbamates andpolyhalogenated sulfenamides and their metabolites and derivatives. Theterm “analyte” also includes combinations of two or more of polypeptidesand proteins, polysaccharides and nucleic acids. Such combinationsinclude, for example, components of bacteria, viruses, chromosomes,genes, mitochondria, nuclei and cell membranes. Protein analytesinclude, for example, immunoglobulins, cytokines, enzymes, hormones,cancer antigens, nutritional markers and tissue specific antigens. Suchproteins include, by way of illustration and not limitation, protamines,histones, albumins, globulins, scleroproteins, phosphoproteins,mucoproteins, chromoproteins, lipoproteins, nucleoproteins,glycoproteins, T-cell receptors, proteoglycans, HLA, unclassifiedproteins, e.g., somatotropin, prolactin, insulin, pepsin, proteins foundin human plasma, blood clotting factors, protein hormones such as, e.g.,follicle-stimulating hormone, luteinizing hormone, luteotropin,prolactin, chorionic gonadotropin, tissue hormones, cytokines, cancerantigens such as, e.g., PSA, CEA, α-fetoprotein, acid phosphatase,CA19.9, CA15.3 and CA125, tissue specific antigens, such as, e.g.,alkaline phosphatase, myoglobin, CPK-MB and calcitonin, and peptidehormones. Other polymeric materials of interest are mucopolysaccharidesand polysaccharides. As indicated above, the term analyte furtherincludes oligonucleotide and polynucleotide analytes such as m-RNA,r-RNA, t-RNA, DNA and DNA-RNA duplexes, for example.

The “MS label precursor” is any molecule that results in an MS label bythe action of the alteration agent. The MS label precursor may itself bean MS label that, through the action of the alteration agent isconverted to another MS label by cleavage, by reaction with a moiety, byderivatization, or by addition or by subtraction of molecules, chargesor atoms, for example, or a combination of two or more of the above.

The term “alteration agent” refers to a substance that has the abilityto alter the MS label precursor. In certain embodiments, alterationagent is able to interact with the MS label precursor to achieve an MSlabel having a unique mass in the range of about 1 Da to about 3 kDa, orin the range of about 1 Da to about 50 Da, or in the range of about 50Da, to about 150 Da, or in the range of about 150 Da to about 700 Da, orin the range of about 700 Da to about 3 kDa. In some examples the uniquemass of the MS label is below about 3 kDa. The MS label precursor can bealtered by bond breaking to form a neutral, negative or positive ion, orradical. The alteration of the MS label precursor by the alterationagent may be by addition of atoms, charges or electrons to, orsubtraction of atoms, charges or electrons from, the MS label precursoror by bond cleavage in, or bond formation in, the MS label precursor.The alteration agents include, but are not limited to, chemical agentssuch as, but not limited to, catalysts (e.g., enzymes (includingpseudoenzymes) and metals), oxidizing agents, reducing agents, acids,bases, agents that promote substitution reactions or replacementreactions; and ionization agents. In some examples, the alteration agentfacilitates the formation of an MS label from the MS label precursor bypromoting the reaction of the MS label precursor with a moiety to formthe MS label, for example. In some examples the alteration agentfacilitates the formation of an MS label from the MS label precursor bypromoting the release of the MS label from the MS label precursor, forexample.

The nature of the MS label precursors may be dependent for example onone or more of the nature of the MS label, the nature of the MS methodemployed, the nature of the MS detector employed, the nature of thetarget rare molecules, the nature of the affinity agent, the nature ofany immunoassay employed, the nature of the sample, the nature of anybuffer employed, the nature of the separation. In some examples, the MSlabel precursors are molecules whose mass can be varied by substitutionand/or chain size. The MS labels produced from the MS label precursorsare molecules of defined mass, which should not be present in the sampleto be analyzed. Furthermore, the MS labels should be in the rangedetected by the MS detector, should not have over-lapping masses andshould be detectable by primary mass. Examples, by way of illustrationand not limitation, of MS label precursors for use in methods of theinvention include, by way of illustration and not limitation,polypeptides, organic and inorganic polymers, fatty acids,carbohydrates, cyclic hydrocarbons, aliphatic hydrocarbons, aromatichydrocarbons, organic carboxylic acids, organic amines, nucleic acids,organic alcohols (e.g., alkyl alcohols, acyl alcohols, phenols, polyols(e.g., glycols), thiols, epoxides, primary, secondary and tertiaryamines, indoles, tertiary and quaternary ammonium compounds, aminoalcohols, amino thiols, phenolic amines, indole carboxylic acids,phenolic acids, vinylogous acid, carboxylic acid esters, phosphateesters, carboxylic acid amides, carboxylic acids from polyamides andpolyesters, hydrazone, oxime, trimethylsilyl enol ether, acetal, ketal,carbamates, ureas, guanidines, isocyanates, sulfonic acids,sulfonamides, sulfonylureas, sulfates esters, monoglycerides, glycerolethers, sphingosine bases, ceramines, cerebrosides, steroids,prostaglandins, carbohydrates, nucleosides and therapeutic drugs.

An MS label precursor can include 1 to about 100,000 MS labels, or about10 to about 100,000 MS labels, or about 100 to about 100,000 MS labels,or about 1,000 to about 100,000 MS labels, or about 10,000 to about100,000 MS labels. The MS label precursor can be comprised of proteins,polypeptides, polymers, particles, carbohydrates, nucleic acids, lipidsor other macromolecules capable of including multiple repeating units ofMS labels by attachment. Multiple MS labels allow amplification as everyMS label precursor can generate many MS labels.

With polypeptide MS label precursors, for example, the chain length ofthe polypeptide can be adjusted to yield an MS label in a mass regionwithout background peaks. Furthermore, MS labels may be produced fromthe MS label precursors having unique masses, which are not present inthe sample tested. The polypeptide MS label precursors can compriseadditional amino acids or derivatized amino acids, which allows methodsto be multiplexed to obtain more than one result at a time. Examples ofpolypeptide MS label precursors include, but are not limited to,polyglycine, polyalanine, polyserine, polythreonine, polycysteine,polyvaline, polyleucine, polyisoleucine, polymethionine, polyproline,polyphenylalanine, polytyrosine, polytryptophan, polyaspartic acid,polyglutamic acid, polyasparagine, polyglutamine, polyhistidine,polylysine and polyarginine, for example. Polypeptide MS labelprecursors differentiated by mixtures of amino acids or derivatizedamino acids generate masses having even or odd election ion with orwithout radicals. In some examples, polypeptides are able to be modifiedby catalysis. For example, by way of illustration and not limitation,phenol and aromatic amines can be added to polythreonine using aperoxidase enzyme as a catalyst. In another example, by way ofillustration and not limitation, electrons can be transferred toaromatic amines using peroxidase enzyme as a catalyst. In anotherexample, by way of illustration and not limitation, phosphates can beremoved from organic phosphates using phosphatases as a catalyst.

In another example, by way of illustration and not limitation, aderivatization agent is employed as a moiety to generate an MS labelfrom an MS label precursor. For example, dinitrophenyl and othernitrophenyl derivatives may be formed from the MS label precursor. Otherexamples include, by way of illustration and not limitation,esterification, acylation, silylation, protective alkylation,derivatization by ketone-base condensations such as Schiff bases,cyclization, formation of fluorescent derivatives, and inorganic anions.The derivatization reactions can occur in microreaction prior to MSanalysis but after affinity reaction or be used to generate MS labelprecursors conjugated to affinity reagents.

In some examples, the MS label precursor can comprise an isotope suchas, but not limited to, ²H, ¹³C, and ¹⁸O, for example, which remains inthe MS label that is derived from the MS label precursor. The MS labelcan be detected by the primary mass or a secondary mass afterionization. In some examples, the MS label precursor is one that has arelatively high potential to cause a bond cleavage such as, but notlimited to, alkylated amines, acetals, primary amines and amides, forexample, where the MS label can generate a mass that has even or oddelection ion with or without radicals. Selection of the polypeptide cangenerate a unique MS spectral signature.

As mentioned above, the alteration agent may be an enzyme (whichincludes pseudoenzymes). In some examples, catalysis can occur withwater insoluble enzyme derivatives immobilized with, for example, silicagels, charcoals, DEAE-cellulose, DEAE-SEPHADEX (cross-linked dextrangel, commercially available from Sigma Aldrich), cellulose citrate,kaolinite, cellulose phosphate, acid clay, AMBERLITE XE-97 (carboxyliccation exchange resin manufactured by Rohm & Haas), carboxymethylcellulose, glass, quartz, dowex-50, starch gel, polyacrylamide gel, polyamino acids, or aminobenzyl cellulose. Cross-linking agents can be usedto immobilize the enzyme. Such cross-linking agents include, but are notlimited to, glutaraldehyde, dimethyl adipimidate, carbodiimide, maleicanhydride, MDA methylenedianiline, hydrazide, and acyl azides, forexample.

In some examples, an enzyme for purposes in accordance with theprinciples described herein is any enzyme with a high turnover rate thatcan convert as an enzyme substrate (such as an MS label precursor) intoan MS label that is detected by the mass detector of a mass spectrometerin the presence of the un-converted substrate. The enzyme should not bein the sample tested or, if present in the sample, it must be removedfrom the sample prior to testing. Examples of enzymes suitable for thispurpose include, but are not limited to, phosphatases (e.g., alkalinephosphatase, lipid phosphatases, tyrosine phosphatase, serinephosphatase, threonine phosphatase, and histidine phosphatase); oxidases(e.g., horse radish peroxidase, copper amine oxidase, D-amino acidoxidase, galactose oxidase, plasma amine oxidase,tryptophan peroxidase,uricase oxidase, and xanthine oxidase); β-galactosidase; transferases(e.g., D-alanine transferase, glycosyl transferase, acyl transferase,alkyl transferase, aryl transferase, single carbon transferase, ketonetransferase, aldehyde transferase, nitrogenous transferase, phosphorustransferase, sulfur transferase, and pentosyl transferase); peptidases(e.g., pepsin, papain, rennin (chymosin), renin, thrombin, trypsin,matrix metallopeptidase, cathespin, cysteine protease, andcarboxypeptidase); aldolases (e.g., carboxyl aldolase, aldehydealdolase, oxo acids, tryptophanase); fatty acid enzymes (e.g., fattyacid amine hydrolase, fatty acid synthase, and cholineacetyltransferase), for example, and combinations of two or more of theabove (e.g., two or more of alkaline phosphatase, acid phosphatase, anoxidase, β-galactosidase, peroxidase, acylase, asparaginase, catalase,chymotrypsin, amylase, glucoamylase, glucose oxidase,glucose-6-phosphate dehydrogenase, hexokinase, invertase, lipase,phosphoglucomutase, ribonuclease, acetylcholinesterase, alcoholdehydrogenase, aldolase, cholinesterase, citrate synthetase, urease,amylglucosidase, carboxypeptidase, cholinesterase, luciferase,ribonuclease, pyruvate kinase, and subtilopeptidase).

Substrates for the enzymes are MS label precursors that comprise an MSlabel that is released by the action of the enzyme on the substrate.Such MS labels that may be part of an enzyme substrate include, by wayof illustration and not limitation, phenols (from substrates such as,for example, p-nitrophenyl phosphate, p-nitrophenyl-β-D-galactoside,amino acids, peptides, carbohydrates (6-phospho-D-gluconate), fattyacids (acetyl-CoA), alkyl amines, glycerols,); and naphthols (fromsubstrates such as, for example, p-nitronaphthyl phosphate,p-nitro-naphthyl-β-D-galactoside); for example.

Metals that may be employed to release an MS label from a moietyattached to an affinity agent include, but are not limited to,transition metals (e.g., palladium, platinum, gold, ruthenium, rhodium,or iridium), chelated metals (e.g., iron, copper, cobalt, magnesiumcomplexed by ethylenediaminetetraacetate (EDTA),N-(2-hydroxyethyl)-ethylenediaminetriacetic acid (HEDTA), ortrans-1,2-cyclohexanediaminetetraacetic acid (CDTA), for example), metaloxidants (e.g., sodium hypochlorite, potassium periodate, silver oxide,chromic acid, potassium permanganate, and sodium perborate) and metalreductants (e.g., lithium aluminum hydride, sodium borohydride, sodiumascorbate, phosphites, and sodium), for example.

The MS label can be detected directly or the released MS label can befurther reacted with another compound to form a derivative MS label,which is detected by MS techniques. A derivative MS label is a compoundthat is formed from an MS label that is obtained from the MS labelprecursor where the compound also is detectable by MS techniques. Thisapproach of forming a derivate MS label further enhances themultiplexing capability of methods in accordance with the principlesdescribed herein. For example, a released phenol or naphthol can coupleto an aromatic amine in the presence of a peroxidase (see, for example,U.S. Pat. No. 5,182,213, the relevant disclosure of which isincorporated herein by reference). In one example, a released naphtholis coupled with a phenylenediamine such as, for example,α-phenylenediamine dihydrochloride, in the presence of a peroxidativelyactive substance in an alkaline medium to produce a derivative MS label.Multiplexing may be achieved using different naphthols and/or differentphenylenediamines.

Internal standards are an important aspect of mass spectral analysis. Insome examples, a second mass label can be added that can be measured (asan internal standard) in addition to the MS label used for detection ofthe rare target molecule. The internal standard has a similar structureto the MS label with a slight shift in mass. The internal standards canbe prepared that comprise additional amino acids or derivatized aminoacids. Alternatively, the internal standard can be prepared byincorporating an isotopic label such as, but not limited to ²H (D), ¹³C,and ¹⁸O, for example. The MS isotope label has a mass higher than thenaturally-occurring substance. For example, the isotope labeled MSlabels, for example, glycerol-C-d7, sodium acetate-C-d7, sodiumpyruvate-C-d7, D-glucose-C-d7, deuterated glucose, and dextrose-C-d7,would serve as internal standards for glycerol, sodium acetate, sodiumpyruvate, glucose and dextrose, respectively.

An MS label precursor or an alteration agent may be attached to anaffinity agent (to yield a modified affinity agent) covalently eitherdirectly by a bond or through the intermediacy of a linking group. Insome embodiments, the preparation of a modified affinity agent may becarried out by employing functional groups suitable for attaching the MSlabel precursor or the alteration agent, to the affinity agent by adirect bond. The nature of the functional groups employed is dependent,for example, on one or more of the nature of the MS label precursor, thenature of the alteration agent, and the nature of the affinity agentincluding the nature of one or more different particles such as, e.g.,carrier particles and label particles that may be part of the affinityagent. A large number of suitable functional groups are available forattaching to amino groups and alcohols; such functional groups include,for example, activated esters including, e.g., carboxylic esters, imidicesters, sulfonic esters and phosphate esters; activated nitrites;aldehydes; ketones; and alkylating agents.

The linking group may be a chain of from 1 to about 60 or more atoms, orfrom 1 to about 50 atoms, or from 1 to about 40 atoms, or from 1 to 30atoms, or from about 1 to about 20 atoms, or from about 1 to about 10atoms, each independently selected from the group normally consisting ofcarbon, oxygen, sulfur, nitrogen, and phosphorous, usually carbon andoxygen. The number of heteroatoms in the linking group may range fromabout 0 to about 8, from about 1 to about 6, or about 2 to about 4. Theatoms of the linking group may be substituted with atoms other thanhydrogen such as, for example, one or more of carbon, oxygen andnitrogen in the form of, e.g., alkyl, aryl, aralkyl, hydroxyl, alkoxy,aryloxy, or aralkoxy groups. As a general rule, the length of aparticular linking group can be selected arbitrarily to provide forconvenience of synthesis with the proviso that there is minimalinterference caused by the linking group with the ability of the linkedmolecules to perform their function related to the methods disclosedherein.

The linking group may be aliphatic or aromatic. When heteroatoms arepresent, oxygen will normally be present as oxy or oxo, bonded tocarbon, sulfur, nitrogen or phosphorous; sulfur will be normally bepresent as thioether or thiono; nitrogen will normally be present asnitro, nitroso or amino, normally bonded to carbon, oxygen, sulfur orphosphorous; phosphorous will be normally bonded to carbon, sulfur,oxygen or nitrogen, usually as phosphonate and phosphate mono- ordiester. Functionalities present in the linking group may includeesters, thioesters, amides, thioamides, ethers, ureas, thioureas,guanidines, azo groups, thioethers, carboxylate and so forth. Thelinking group may also be a macro-molecule such as polysaccharides,peptides, proteins, nucleotides, and dendrimers.

In some embodiments the MS label precursor, or the alteration agent, asthe case may be, and the affinity agent may be linked togethernon-covalently. Members of a binding pair, usually a specific bindingpair, are employed where one member is linked to the affinity agent andthe other member is linked to the MS label precursor or to thealteration agent. Binding of the binding pair members results in thenon-covalent linking of the affinity agent and the MS label precursor orthe alteration agent. The binding pair members may be linked directly toone or both of the MS label precursor, or the alteration agent, and theaffinity agent or indirectly through the intermediacy of a linkinggroup, the nature of which is discussed above. In some examples, themembers of the specific binding pair have a relatively high bindingconstant such as, by way of illustration and not limitation, avidin(streptavidin)-biotin binding, fluorescein (FITC) and antibody for FITC,rhodamine (Texas red) and antibody for rhodamine, digitonin (DIG) andantibody for DIG, non-human species antibody (e.g., goat, rabbit, mouse,chicken, sheep) and anti-species antibody, for example.

The modified affinity agents can be prepared by linking each differentaffinity agent in separate, individual reactions to the MS labelprecursor or the alteration agent and then combining the modifiedaffinity agents to form a mixture comprising the modified affinityagents. Alternatively, the different affinity agents can be combined andthe reaction to link the affinity agents to the MS label precursor orthe alteration agent can be carried out on the combination. This allowsthe method to be multiplexed for more than one result at a time.

An amount of each different modified affinity agent that is employed inthe methods of the invention is dependent for example on one or more ofthe nature and potential amount of each different population of targetrare molecules, the nature of the MS label, the nature of the affinityagent, the nature of a cell if present, the nature of a particle ifemployed, and the amount and nature of a blocking agent if employed. Insome examples, the amount of each different modified affinity agentemployed is about 0.001 μg/μL to about 100 μg/μL, or about 0.001 μg/μLto about 80 μg/μL, or about 0.001 μg/μL to about 60 μg/μL, or about0.001 μg/μL to about 40 μg/μL, or about 0.001 μg/μL to about 20 μg/μL,or about 0.001 μg/μL to about 10 μg/μL, or about 0.5 μg/μL to about 100μg/μL, or about 0.5 μg/μL to about 80 μg/μL, or about 0.5 μg/μL to about60 μg/μL, or about 0.5 μg/μL to about 40 μg/μL, or about 0.5 μg/μL toabout 20 μg/μL, or about 0.5 μg/μL to about 10 μg/μL.

The number of alteration agents employed may be one per MS labelprecursor, or one per two MS label precursors, or one per three MS labelprecursors up to one per all MS label precursors employed depending onone or more of the nature of the MS label precursor, the nature of thealteration agent, whether the alteration agent is free in the medium orpart of a modified affinity agent, and the nature and number ofdifferent affinity reagents used. For example, where each of the MSlabel precursors include a labile ester or a labile amide linkage ofdifferent MS labels to the affinity agents, a single alteration agentmay be employed that results in hydrolysis of the disulfide, ester oramide linkages to yield the different MS labels. In other examplesutilizing one alteration agent, or fewer alteration agents than thenumber of MS label precursors, may be employed. In another example, adifferent alteration agent can be used to generate an MS label for eachdifferent type of affinity agent used.

The combination comprising the sample (optionally concentrated) and themodified affinity agents in the aqueous medium is treated by holding fora period of time and at a temperature for binding of the modifiedaffinity agents to target rare molecules on the cells or on the particlereagents. For each modified affinity agent that comprises an alterationagent, an MS label precursor upon which the alteration agent acts isincluded in the combination wherein the MS label precursor is convertedto the MS label. In some examples, an additional moiety is added wherethe alteration agent facilitates the reaction of the moiety with the MSlabel precursor to yield an MS label. In some examples, the modifiedaffinity agent comprises an MS label precursor and the alteration agentis included in the combination as an unbound substance in the medium. Inthis example, the alteration agent acts upon the MS label precursor ofthe affinity agent to produce an MS label. In some examples, a firstalteration agent is employed that releases an entity that comprises anMS label precursor from the affinity agent and a second alteration agentis subsequently employed to facilitate the formation of an MS label froman MS label precursor.

The temperature and duration of this treatment is dependent for exampleon the nature of the sample, the nature of the target rare molecules,the nature of the non-rare molecules, the nature of the modifiedaffinity agents, the nature of the MS label precursors, and the natureof the alteration agents. In some examples, moderate temperatures arenormally employed and usually constant temperature, preferably, roomtemperature. Temperatures during holding a period normally range fromabout 5° C. to about 99° C. or from about 15° C. to about 70° C., orabout 20° C. to about 45° C., for example. The holding period is about0.2 seconds to about 24 hours, or about 1 second to about 6 hours, orabout 2 seconds to about 1 hour, or about 1 to about 15 minutes, forexample. The time period depends on, for example, the temperature of themedium and the rate of binding of the various reagents.

Modified affinity agents, i.e., affinity agents that have been actedupon by an alteration agent, which have become bound to target raremolecules, optionally, are separated from modified affinity agents thathave not become bound to target molecules. In some examples, thisseparation involves reducing the number of non-rare molecules in thesample.

Contact of the treated sample with the essentially non-absorbentmembrane is continued for a period of time sufficient to achieveretention of the target rare cells or the particle-bound target raremolecules on a surface of the essentially non-absorbent membrane toobtain a surface of the essentially non-absorbent membrane havingdifferent populations of target rare cells or the particle-bound targetrare molecules as discussed above. The period of time may be dependentfor example on one or more of the nature and size of the differentpopulations of target rare cells or particle-bound target raremolecules, the nature of the porous matrix, the size of the pores of theporous matrix, the level of vacuum applied to the blood sample on theporous matrix, the volume to be filtered, and the surface area of theporous matrix. In some examples, the period of contact is about 1 minuteto about 1 hour, about 5 minutes to about 1 hour, or about 5 minutes toabout 45 minutes, or about 5 minutes to about 30 minutes, or about 5minutes to about 20 minutes, or about 5 minutes to about 10 minutes, orabout 10 minutes to about 1 hour, or about 10 minutes to about 45minutes, or about 10 minutes to about 30 minutes, or about 10 minutes toabout 20 minutes, for example.

The retentate is subjected to a second alteration agent that facilitatesthe formation of an MS label from the MS label precursor from theaffinity agent if the first alteration agent does not facilitate theformation of an MS label from the MS label precursor.

The retentate is subjected to MS analysis to determine the presenceand/or amount of each different MS label. The presence and/or amount ofeach different MS label are related to the present and/or amount of eachdifferent population of target rare cells and/or particle-bound targetrare molecules.

MS analysis determines the mass-to-charge ratio (m/z) of molecules foraccurate identification and measurement. The MS method may ionize themolecules into masses as particles by several techniques that mayinclude, but are not limited to, atmospheric pressure chemicalionization (APCI), electrospray ionization (ESI), inductive electrosprayionization (iESI), chemical ionization (CI), and electron ionization(EI), fast atom bombardment (FAB), field desorption/field ionization(FC/FI), thermospray ionization (TSP), nanospray ionization, forexample. The masses are filtered and separated in the mass detector byseveral techniques that include, by way of illustration and notlimitation, Time-of-Flight (TOF), ion traps, quadrupole mass filters,sector mass analysis, multiple reaction monitoring (MRM), and Fouriertransform ion cyclotron resonance (FTICR). The MS method detects themolecules using, for example, a microchannel plate, electron multiplier,or Faraday cup. The MS method can be repeated as a tandem MS/MS method,in which charged mass particles from a first MS are separated into asecond MS.

Mass analyzers include, but are not limited to, quadrupoles,time-of-flight (TOF) analyzers, magnetic sectors, Fourier transform iontraps, and quadrupole ion traps, for example. Tandem (MS-MS) massspectrometers are instruments that have more than one analyzer. Tandemmass spectrometers include, but are not limited to,quadrupole-quadrupole, magnetic sector-quadrupole,quadrupole-time-of-flight, for example. The detector of the massspectrometer may be, by way of illustration and not limitation, aphotomultiplier, an electron multiplier, or a micro-channel plate, forexample.

Following the analysis by mass spectrometry, the presence and/or amountof each different mass spectrometry label is related to the presentand/or amount of each different population of target rare cells and/orthe particle-bound target rare molecules. The relationship between theMS label and a target molecule is established by the modified affinityagent employed, which is specific for the target molecule. Calibratorsare employed to establish a relationship between an amount of signalfrom an MS label and an amount of target rare molecules in the sample.The samples may be subjected to further analysis.

As mentioned above, the essentially non-absorbent membrane may comprisemore than one pore and the electrical field may be activated toselectively release droplets from an individual pore. The releaseddroplets are subjected to mass spectrometry analysis to determine anarea adjacent the individual pore where a particular MS label islocated. The liquid on the membrane corresponding to the area is removedfor analysis. The liquid adjacent the individual pore may be removed byany of the methods mentioned above. Methods for analysis include, butare not limited to immunoassays, enzyme amplification, cell filtration,nucleic acid sequencing, mass analysis, chemical analysis, nucleic acidamplification, nucleic acid expression, cell growth and cellularresponse assays, for example, or combinations of two or more thereof.

In one example, sample is collected into a container with a suitablecell buffer. The collected sample is subjected to filtration toconcentrate the number of cell-bound target rare molecules over that ofother molecules in the sample such as, for example, non-rare cells. Anaffinity agent that comprises an alteration agent linked to an antibodythat is specific for the cell-bound target rare molecule is combinedwith the concentrated sample retained on an essentially non-absorbentmembrane of a filtration device. After a suitable incubation period, themembrane is washed with a buffer. An MS label precursor is added to thesample on the membrane. The alteration agent of the affinity agent ispart of an immune complex comprising the affinity agent and thecell-bound target molecule. If the target rare molecule is present inthe sample, the alteration agent acts upon the MS label precursor toproduce an MS label that corresponds to the target rare molecule. Theessentially non-absorbent membrane is subjected to an electric field andthe MS label is collected. In some embodiments, spray liquid or spraysolvent is added to a well comprising the essentially non-absorbentmembrane, which is exposed to the electrical field, and optionally avacuum, to release droplets of the liquid from the porous membrane. Ifthe target rare molecule is present in the sample, the MS label willgive a distinctive spectrum that corresponds to the target raremolecule. This spectrum can be correlated to concentration and theposition of the rare cell on the membrane. The position of the rare cellon the membrane can identify where to remove the rare cell for furtheranalysis. In the example above, detection of only one target raremolecule is depicted; however, it is to be appreciated that any numberof target rare molecules may be determined in a single method on asingle sample using various MS label precursors as discussed above asdiscussed above.

In another example, sample is collected into a container with a suitablecell buffer. The collected sample is subjected to filtration toconcentrate the number of cell-bound target rare molecules over that ofother molecules in the sample such as, for example, non-rare cells. Anaffinity agent that comprises an MS label precursor linked to anantibody that is specific for the cell-bound target rare molecule iscombined with the concentrated sample retained on an essentiallynon-absorbent membrane of a filtration device. After a suitableincubation period, the membrane is washed with a buffer. An alterationagent is added to the sample on the membrane. The MS label precursor ofthe affinity agent is part of an immune complex comprising the affinityagent and the cell-bound target molecule. If the target rare molecule ispresent in the sample, the alteration agent acts upon the MS labelprecursor to produce an MS label that corresponds to the target raremolecule. The essentially non-absorbent membrane is subjected to anelectric field and the MS label is collected. In some embodiments, sprayliquid or spray solvent is added to a well comprising the essentiallynon-absorbent membrane, which is exposed to the electrical field, andoptionally a vacuum, to release droplets of the liquid from the porousmembrane. If the target rare molecule is present in the sample, the MSlabel will give a distinctive spectrum that corresponds to the targetrare molecule. This spectrum can be correlated to concentration and theposition of the rare cell on the membrane. In the above example,detection of only one target rare molecule is depicted; however, it isto be appreciated that any number of target rare molecules may bedetermined in a single method on a single sample using various MS labelprecursors as discussed above as discussed above.

In another example, sample is collected into a container and added tothe essentially non-absorbent membrane in diluted or undiluted form. Inthis example, the target rare molecule is non-particulate, i.e., thetarget rare molecule is not bound to a cell or other particle. Thecollected sample is combined with a particle reagent that comprises aparticle to which is attached an antibody for the target rare molecule.After an incubation period to permit binding of the non-cell-boundtarget rare molecule to the antibody on the particle to giveparticle-bound non-cell-bound target rare molecule, the sample issubjected to filtration to concentrate the number of particle-boundnon-cell-bound target rare molecules over that of other molecules in thesample such as, for example, non-rare cells. Sample retained on thesurface of the filtration device is washed with a suitable buffer. Anaffinity agent that comprises an alteration agent linked to an antibodythat is specific for the particle-bound non-cell-bound target raremolecule is combined with the concentrated sample retained on a membraneof a filtration device. After a suitable incubation period, the membraneis washed with a buffer. An MS label precursor is added to the sample onthe membrane. The alteration agent of the affinity agent is part of animmune complex comprising the affinity agent and the particle-boundnon-cell-bound target molecule. If the target rare molecule is presentin the sample, the alteration agent acts upon the MS label precursor toproduce an MS label that corresponds to the target rare molecule. Theessentially non-absorbent membrane is subjected to an electric field andthe MS label is collected. In some embodiments, spray liquid or spraysolvent is added to a well comprising the essentially non-absorbentmembrane, which is exposed to the electrical field, and optionally avacuum, to release droplets of the liquid from the porous membrane. Ifthe target rare molecule is present in the sample, the MS label willgive a distinctive spectrum that corresponds to the target raremolecule. This spectrum can be correlated to concentration and theposition of the rare cell on the membrane. In the above example,detection of only one non-cell-bound target rare molecule is depicted;however, it is to be appreciated that any number of target raremolecules (both cell-bound and non-cell bound) may be determined in asingle method on a single sample using various MS label precursors asdiscussed above.

In another example, liquid sample is collected into a container andadded to the essentially non-absorbent membrane in diluted or undilutedform. In this example, the target rare molecule is non-particulate,i.e., the target rare molecule is not bound to a cell or other particle.The collected sample is combined with a particle reagent that comprisesa particle to which is attached an antibody for the target raremolecule. After an incubation period to permit binding of thenon-cell-bound target rare molecule to the antibody on the particle togive particle-bound non-cell-bound target rare molecule, the sample issubjected to filtration to concentrate the number of particle-boundnon-cell-bound target rare molecules over that of other molecules in thesample such as, for example, non-rare cells. Sample retained on thesurface of the filtration device is washed with a suitable buffer. Anaffinity agent that comprises an MS label precursor linked to anantibody that is specific for the particle-bound non-cell-bound targetrare molecule is combined with the concentrated sample retained on amembrane of a filtration device. After a suitable incubation period, themembrane is washed with a buffer. An alteration agent is added to thesample on the membrane. The MS label precursor of the affinity agent ispart of an immune complex comprising the affinity agent and theparticle-bound non-cell-bound target molecule. If the target raremolecule is present in the sample, the alteration agent acts upon the MSlabel precursor to produce an MS label that corresponds to the targetrare molecule. The essentially non-absorbent membrane is subjected to anelectric field and the MS label is collected. In some embodiments, sprayliquid or spray solvent is added to a well comprising the essentiallynon-absorbent membrane, which is exposed to the electrical field, andoptionally a vacuum, to release droplets of the liquid from the porousmembrane. If the target rare molecule is present in the sample, the MSlabel will give a distinctive spectrum that corresponds to the targetrare molecule. This spectrum can be correlated to concentration and theposition of the rare cell on the membrane. In the example above,detection of only one non-cell-bound target rare molecule is depicted;however, it is to be appreciated that any number of target raremolecules (both cell-bound and non-cell bound) may be determined in asingle method on a single sample using various MS label precursors asdiscussed above.

In another example, sample is collected into a container and added tothe essentially non-absorbent porous membrane in dilute or undilutedform. In this example, the target rare molecule is non-particulate,i.e., the target rare molecule is not bound to a cell or other particle.The collected sample is combined with a particle reagent that comprisesa particle to which is attached an antibody for the target raremolecule. After an incubation period to permit binding of thenon-cell-bound target rare molecule to the antibody on the particle togive particle-bound non-cell-bound target rare molecule, the sample issubjected to filtration to concentrate the number of particle-boundnon-cell-bound target rare molecules over that of other molecules in thesample such as, for example, non-rare cells. Sample retained on thesurface of the filtration device is washed with a suitable buffer. Analteration agent is added to the sample on the membrane that convertsthe non-cell-bound target rare molecules to a MS label. After a suitableincubation period, the membrane is washed with a buffer. If the targetrare molecule is present in the sample, the alteration agent acts uponthe target rare molecule to produce an MS label that corresponds to thetarget rare molecule. The essentially non-absorbent membrane issubjected to the charge field and the MS labels collected. In someembodiments, spray liquid or spray solvent is added to a well comprisingthe essentially non-absorbent membrane, which is exposed to theelectrical field, and optionally a vacuum, to release droplets of theliquid from the porous membrane. If the target rare molecule is presentin the sample, the MS label will give a distinctive spectrum thatcorresponds to the target rare molecule. This spectrum can be correlatedto concentration and the position of the rare cell on the membrane. Inthe example above, detection of only one non-cell-bound target raremolecule is depicted; however, it is to be appreciated that any numberof target rare molecules (both cell-bound and non-cell bound) may bedetermined in a single method on a single sample using various MS labelprecursors as discussed above.

Examples of Methods Employing Particle Amplification

As mentioned above, in one approach, particle amplification is utilizedand provides for the aggregation or clustering particles to formparticle aggregates that comprise MS labels or MS label precursors.

The phrase “particle amplification” refers to the formation ofaggregates or clusters of particles in which a number of label particlesindicative of a single target rare molecule are enhanced. In someexamples, the number of label molecules in a particle aggregate that isindicative of a target rare molecule are 10¹⁰ to 1, or 10⁹ to 1, or 10⁸to 1, or 10⁷ to 1, or10⁶ to 1, or 10⁵ to 1, or 10⁴ to 1, or 10³ to 1, or10² to 1, or 10 to 1, or 10¹⁰ to 10², or 10¹⁰ to 10³, or 10¹⁰ to 10⁴, or10¹⁰ to 10⁵. Particle amplification is achieved by employing a largerparticle (carrier particle) associated with many smaller label particlesthat have many MS labels or MS label precursors associated therewith.

The term “associated with” refers to the manner in which two moietiesare bound to one another. The association may be through covalent ornon-covalent binding as defined above. The attachment may beaccomplished by a direct bond between the two moieties or a linkinggroup can be employed between the two moieties. Linking groups may be,for example, as described above.

The composition of the carrier particle may be, for example, asdescribed above for capture particle entities. The size of the carrierparticle is large enough to accommodate one or more label particles. Theratio of label particles to a single carrier particle may be for example10⁶ to 1, or 10⁵ to 1, or 10⁴ to 1, or 10³ to 1, or 10² to 1, or 10 to1. The diameter of the carrier particle may also be dependent forexample on one or more of the nature of the target rare molecule, thenature of the sample, the nature and the pore size of an essentiallynon-absorbent membrane, the adhesion of the particle to membrane, thesurface of the particle, the surface of the membrane, the liquid ionicstrength, liquid surface tension and components in the liquid, and thenumber, size, shape and molecular structure of associated labelparticles. When a porous matrix is employed in a filtration separationstep, the diameter of the carrier particles should be large enough tohold a number of label particles to achieve the benefits of particleamplification but small enough to be pass through the pores of anessentially non-absorbent membrane of a filtration device. In someexamples, the average diameter of the carrier particles should be atleast about 0.1 microns and not more than about 1 micron, or not morethan about 5 microns. In some examples, the carrier particles have anaverage diameter from about 0.1 microns to about 5 microns, or about 1micron to about 3 microns, or about 4 microns to about 5 microns, about0.2 microns to about 0.5 microns, or about 1 micron to about 3 microns,or about 4 microns to about 5 microns.

The composition of the label particle may be, for example, as describedabove for capture particle entities. The size of the label particles maybe dependent for example on one or more of the nature and size of thecarrier particle, the nature and size of the MS label, or the MS labelprecursor, of the alteration agent, the nature of the target raremolecule, the nature of the sample, the nature and the pore size of theessentially non-absorbent membrane, the surface of the particle, thesurface of the membrane, the liquid ionic strength and, liquid surfacetension and components in the liquid. In some examples, the averagediameter of the label particles should be at least about 0.01 micronsand not more than about 0.1 microns, or not more than about 1 micron. Insome examples, the label particles have an average diameter from about0.01 microns to about 1 micron, or about 0.01 microns to about 0.5microns, or about 0.01 microns to about 0.4 microns, or about 0.01microns to about 0.3 microns, or about 0.01 microns to about 0.2microns, or about 0.01 microns to about 0.1 microns, or about 0.01microns to about 0.05 microns, or about 0.1 microns to about 0.5microns, or about 0.05 microns to about 0.1 microns. In some examples,the label particle may be a silica nanoparticle, which can be linked tomagnetic carrier particles that have free carboxylic acid groups byionic association.

The number of MS labels or MS label precursors associated with the labelparticle may be dependent for example on one or more of the nature andsize of the MS label or MS label precursor, the nature and size of thelabel particle, the nature of the linker arm, the number and type offunctional groups on the label particle, and the number and type offunctional groups on the MS label precursor, for example. In someexamples, the number of MS labels or MS label precursors associated witha single label particle is about 10⁷ to 1, or about 10⁶ to 1, or about10⁵ to 1, or about 10⁴ to 1, or about 10³ to 1, or about 10² to 1, orabout 10 to 1.

As mentioned above, some examples are directed to methods of one or moredifferent populations of target rare molecules in a sample suspected ofcontaining the one or more different populations of rare molecules andnon-rare molecules. The sample that has an enhanced concentration of theone or more different populations of target rare molecules over that ofthe non-rare molecules wherein the target rare molecules are inparticulate form is incubated with, for each different population oftarget rare molecules, an affinity agent that comprises a bindingpartner that is specific for and binds to a target rare molecule of oneof the populations of the target rare molecules. The affinity agentcomprises an MS label precursor or a first alteration agent. For eachdifferent population of target rare molecules, the affinity agentcomprises a particle reagent. The first alteration agent facilitates theformation of an MS label from the MS label precursor or releases anentity that comprises the MS label precursor from the affinity agent.During the incubating, for each different population of target raremolecules, particle aggregates are formed from the particle reagent ofthe affinity agent. A retentate and a filtrate are formed by contactingthe incubated samples with an essentially non-absorbent membrane. Theretentate becomes disposed on the essentially non-absorbent membrane.Spray liquid or spray solvent is added to a well comprising theessentially non-absorbent membrane, which is exposed to an electricalfield, and optionally a vacuum, to release droplets of the liquid fromthe porous membrane.

In some examples, vacuum is applied to the sample on the essentiallynon-absorbent membrane to facilitate passage of the liquid dropletsthrough the pores of the essentially non-absorbent membrane. The levelof vacuum applied may be dependent for example on one or more of thenature and size of the different populations of rare cells and/orparticle reagents, the nature of the essentially non-absorbent membrane,and the size of the pores of the essentially non-absorbent membrane. Insome examples, the level of vacuum applied is about 1 millibar to about100 millibar, or about 1 millibar to about 80 millibar, or about 1millibar to about 50 millibar, or about 1 millibar to about 40 millibar,or about 1 millibar to about 30 millibar, or about 1 millibar to about25 millibar, or about 1 millibar to about 20 millibar, or about 1millibar to about 15 millibar, or about 1 millibar to about 10 millibar,or about 5 millibar to about 100 millibar, or about 5 millibar to about80 millibar, or about 5 millibar to about 50 millibar, or about 5millibar to about 30 millibar, or about 5 millibar to about 25 millibar,or about 5 millibar to about 20 millibar, or about 5 millibar to about15 millibar, or about 5 millibar to about 10 millibar. The applicationof vacuum is coordinated with application of the electrical field so theliquid droplets can be selectively released from individual microwellscomprising an essentially non-absorbent membrane in accordance with theprinciples described herein.

The droplets are subjected to MS analysis to determine the presenceand/or amount of each different MS label. The presence and/or amount ofeach different MS label is related to the present and/or amount of eachdifferent population of non-cellular target rare molecules in thesample. In this manner samples may be identified for further analysis.In one approach, the essentially non-absorbent membrane containingmaterial of interest may be removed by any convenient method. Examplesof such methods include, but are not limited to, punching out theportion of the essentially non-absorbent membrane of interest or byfiltration, for example.

The size of the particle aggregates is dependent on one or more of thenature and size of the capture particle, the nature and size of thecarrier particle, the nature and size of the label particle, the natureand size of the linking groups, the nature and size of the MS label orthe MS label precursor, the nature of the alteration agent, the natureof the target rare molecule, the nature of the sample, the nature andthe pore size of a filtration matrix, the surface of the particle, thesurface of the matrix, the liquid ionic strength and, liquid surfacetension and components in the liquid, for example. In some examples inaccordance with the principles described herein, the average diameter ofthe particle aggregates is at least about 0.1 microns and not more thanabout 500 microns, or not more than about 1,000 microns. In someexamples, the particle aggregates have an average diameter from about0.1 microns to about 1,000 microns, or about 0.1 microns to about 500microns, or about 0.1 microns to about 100 microns, or about 0.1 micronsto about 10 microns, or about 0.1 microns to about 5 microns, or about0.1 microns to about 1 micron, or about 1 micron to about 10 microns, orabout 10 microns to about 500 microns, or about 10 microns to about 100microns, for example.

In one example, the target rare molecule is attached to the surface of acell on the order of about 10 microns (m). Carrier particles having anaverage diameter of about 1 μm in this example are linked by means of afirst linking group to a specific binding partner such as, for example,an antibody for the target rare molecule. A second linking group linksadditional carrier particles to one another in a linear manner. In thisexample, the number of carrier particles per cell is about 1,000.Furthermore, there are approximately 100 label particles (about 200 nmin diameter) per each carrier particle linked thereto by means of athird linking group. For each label particle there are about 10⁵ MSlabels (Mass labels) linked thereto by means of a fourth linking group.In this example, the MS labels have a size of about 1 nm. The linkinggroups may be chosen from any linking group as described above and twoor more thereof may be the same or each of the linking groups may bedifferent from one another. In some examples, one or more of the linkinggroups have a cleavable moiety so that, for example, carrier particlesmay be cleaved from one another or from the cell and/or label particlesmay be cleaved from the carrier particles, and/or MS labels or MS labelprecursors may be cleaved from the label particles. Cleavage of thevarious linking groups may be carried out sequentially where thecleavable moieties of the linking groups differ from one another.

As mentioned above, one or more linking groups may comprise a cleavablemoiety that is cleavable by a cleavage agent. The nature of the cleavageagent is dependent on the nature of the cleavable moiety. Cleavage ofthe cleavable moiety may be achieved by chemical or physical methods,involving one or more of oxidation, reduction, solvolysis, e.g.,hydrolysis, photolysis, thermolysis, electrolysis, sonication, andchemical substitution, for example. Examples of cleavable moieties andcorresponding cleavage agents, by way of illustration and notlimitation, include disulfide that may be cleaved using a reducingagent, e.g., a thiol; diols that may be cleaved using an oxidationagent, e.g., periodate; diketones that may be cleaved by permanganate orosmium tetroxide; diazo linkages or oxime linkages that may be cleavedwith hydrosulfite; β-sulfones, which may be cleaved under basicconditions; tetralkylammonium, trialkylsulfonium, tetralkylphosphonium,where the a-carbon is activated, e.g., with carbonyl or nitro, that maybe cleaved with base; ester and thioester linkages that may be cleavedusing a hydrolysis agent such as, e.g., hydroxylamine, ammonia ortrialkylamine (e.g., trimethylamine or triethylamine) under alkalineconditions; quinones where elimination occurs with reduction;substituted benzyl ethers that can be cleaved photolytically; carbonatesthat can be cleaved thermally; metal chelates where the ligands can bedisplaced with a higher affinity ligand; thioethers that may be cleavedwith singlet oxygen; hydrazone linkages that are cleavable under acidicconditions; quaternary ammonium salts (cleavable by, e.g., aqueoussodium hydroxide); trifluoroacetic acid-cleavable moieties such as,e.g., benzyl alcohol derivatives, teicoplanin aglycone, acetals andthioacetals; thioethers that may be cleaved using, e.g., HF or cresol;sulfonyls (cleavable by, e.g., trifluoromethane sulfonic acid,trifluoroacetic acid, or thioanisole); nucleophile-cleavable sites suchas phthalamide (cleavable, e.g., with substituted hydrazines); ionicassociation (attraction of oppositely charged moieties) where cleavagemay be realized by changing the ionic strength of the medium, adding adisruptive ionic substance, lowering or raising the pH, adding asurfactant, sonication, and adding charged chemicals; and photocleavalbebonds that are cleavable with light having an appropriate wavelengthsuch as, e.g., UV light at 300 nm or greater.

In one example, a cleavable linkage may be formed using conjugation withN-succinimidyl 3-(2-pyridyldithio)propionate) (SPDP), which comprises adisulfide bond. For example, a label particle comprising an aminefunctionality is conjugated to SPDP and the resulting conjugate can thenbe reacted with a MS label comprising a thiol functionality, whichresults in the linkage of the MS label moiety to the conjugate. Adisulfide reducing agent (such as, for example, dithiothreitol (DTT) ortris(2-carboxyethyl)phosphine (TCEP)) may be employed as an alterationagent to release a thiolated peptide as an MS label.

An example, by way of illustration and not limitation, of the formationof a particle aggregate (particle cluster) on a membrane of a filtrationdevice is discussed next. A cell or a capture particle that has captureda non-particulate target rare molecule in a sample is contacted with amembrane of a filtration slide, wherein the size of the pores of themembrane are as described above for retaining cells or particle-boundtarget rare molecules. After suitable washing to remove non-particulatematerial and to reduce the number of non-rare molecules and non-rarecells as discussed above, a set of carrier particles as described aboveis added for each different population of target rare molecules whereeach set of the carrier particles comprise a specific binding partnerspecific for a different target rare molecule to be determined. Thespecific binding partner is linked to the carrier particle by means of afirst linking group. Carrier particles are linked to one anotheremploying a second linking group. After another washing step, labelparticles are added where each set of the label particles comprise an MSlabel or an MS label precursor for a different target rare molecule tobe determined. The label particles comprise a functionality that isreactive with a functionality on the carrier particles. The reaction ofthe functionalities provides for the formation of a third linking group.The MS labels or the MS label precursors are bound to the labelparticles by means of a fourth linking group. As a result, a particlecluster is formed comprising the target rare molecule, the carrierparticles, the label particles and the MS labels or MS label precursors.

In some examples, one or more of the linking groups are formedcovalently as described above employing appropriate correspondingfunctionalities of functional groups as discussed above. In someexamples, one of more of the linking groups is formed non-covalently asdiscussed above. Members of a binding pair, usually a specific bindingpair, are employed where one member is linked to one linking groupmoiety and the other member is linked to a second linking group moiety.When the binding pair members bind, the linking group is formed thatincludes the binding pair members and the two linking group moieties.Binding of the binding pair members results in the non-covalent linkingof the two linking group moieties that ultimately form the linkinggroup. The linking group moieties may be a bond or a linking group asdiscussed above. As mentioned above, the members of the binding pairhave a relatively high binding constant such as, by way of illustrationand not limitation, avidin (streptavidin)-biotin binding, fluorescein(FITC) and antibody for FITC, rhodamine (Texas red) and antibody forrhodamine, digitonin (DIG) and antibody for DIG, non-human speciesantibody (e.g., goat, rabbit, mouse, chicken, sheep) and anti-speciesantibody, for example.

In some examples, by way of illustration and not limitation, the firstlinking group may involve a non-cleavable bond employing a secondaryantibody linked to biotin where the secondary antibody binds to theantibody for the target rare molecule and the biotin binds tostreptavidin molecules on the surface of a carrier particle.Alternatively, the antibody can be directly conjugated to the carrierparticle through amide bounds to the carboxylic acids on the particleand amines on the antibody using commonly known bioconjugation methods.In another example, the first linking group may involve a cleavablelinkage employing a small molecule peptide linked to biotin and attachedto the antibody by a disulfide linker made by reaction with, forexample, SPDP. In some examples, the second linking group may include anon-cleavable linkage where the carrier particle has streptavidinmolecules on its surface and a conjugate of biotin and a small moleculesuch as, for example, biotin-FITC, is employed to form the linkinggroup. When a cleavable linkage is desired for the second linking group,the biotin-FITC agent includes a cleavable moiety such as, for example,a disulfide bond. The small molecule portion, e.g., FITC portion, of thesecond linking group binds to a binding partner for the small molecule(e.g., an antibody for FITC) on the surface of the carrier particle. Thethird linking group may include a non-cleavable linkage where thelinking moiety has a peptide attached to FITC or biotin by an amide bondor the third linking group may include a cleavable linkage where thelinking moiety has a peptide attached to FITC or biotin by a disulfidebond. The third linking group may include an ionic linkage where theionized amines or other groups on the label particle are attracted tothe ionized carboxylic acid or other groups on the label particle. Asexplained above, an MS label or MS label precursor is attached to alabel particle by a cleavable bond such as, but not limited to, apeptide or other MS label attached by a disulfide bond.

The phrase “small molecule” refers to a molecule having a molecularweight in the range of about 100 to about 2,000, or about 200 to about2,000, or about 300 to about 2,000, or about 500 to about 2,000, orabout 1,000 to about 2,000, or about 500 to about 1,500, or about 1,000to about 1,500, or about 1,000 to about 1,200, for example. Examples ofsmall molecules, by way of illustration and not limitation, includebiotin, digoxin, digoxigenin, 2,4-dinitrophenyl, fluorescein, rhodamine,small peptides (meeting the aforementioned molecular weight limits),vitamin B12 and folate, for example. Examples of small molecule-bindingpartner for the small molecule pairs, by way of illustration and notlimitation, include biotin-binding partner for biotin (e.g., avidin,streptavidin and antibody for biotin), digoxin-binding partner fordigoxin (e.g., antibody for digoxin), digoxigenin-binding partner fordigoxigenin (e.g., antibody for digoxigenin), 2,4-dinitrophenyl andbinding partner for 2,4-dinitrophenyl (e.g., antibody for2,4-dinitrophenyl), fluorescein-binding partner for fluorescein (e.g.,antibody for fluorescein), rhodamine-binding partner for rhodamine(e.g., antibody for rhodamine), peptide-binding partner for the peptide(e.g., antibody for the peptide), analyte-specific binding partners(e.g., intrinsic factor for B12, folate binding factor for folate), forexample.

Examples of small molecule peptides, which may function also as MSlabels, include, by way of illustration and not limitation, peptidesthat comprise two or more of histidine, lysine, phenylalanine, leucine,alanine, methionine, asparagine, glutamine, aspartic acid, glutamicacid, tryptophan, proline, valine, tyrosine, glycine, threonine, serine,arginine, cysteine and isoleucine and derivatives thereof. In someexamples, the peptides have a molecular weight of about 100 to about3,000 mass units and may contain 3 to 30 amino acids. In some examples,the peptides comprise nine amino acids selected from the groupconsisting of tyrosine, glycine, methionine, threonine, serine,arginine, phenylalanine, cysteine and isoleucine and have masses of1,021.2; 1,031.2; 1,033.2; 1,077.3; 1,087.3; 1,127.3; 1,137 mass units;or 3 amino acids from the above group and having masses of 335.4, 433.3,390.5, 426.5, and 405.5 mass units. The number of amino acids in thepeptide is determined by, for example, the nature of the MS techniqueemployed. For example, when using MALDI for detection, the peptide canhave a mass in the range of about 600 to about 3,000 and is constructedof about 6 to about 30 amino acids. Alternatively, when using EIS fordetection, the peptide has a mass in the range of about 100 to about1,000 and is constructed of 1 to 9 amino acids or derivatives of, forexample. In some examples, the number of amino acids in the peptidelabel may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, for example.

The use of peptides as MS labels has several advantages, which include,but are not limited to, the following: 1) relative ease of conjugationto proteins, antibodies, particles and other biochemical entities; 2)relative ease with which the mass can be altered to allow many differentmasses thus providing for multiplexed assay formats and standards; and3) adjustability of the mass to a mass spectrometer used. Forconjugation, the peptides can have a terminal cysteine that is employedin the conjugation. For ionization, the peptides can have charged aminegroups. In some examples, the amino acid peptides have N-terminal freeamine and C-terminal free acid. In some examples, the amino acidpeptides are isotope labeled or derivatized with an isotope. Thepeptides may be conjugated to a small molecule such as, for example,biotin or fluorescein, for binding to a corresponding binding partnerfor the small molecule, which in this example is streptavidin orantibody for fluorescein. Biotin or fluorescein may be conjugated at theN-terminal with the C-terminal being free acid.

The methods described herein involve trace analysis, i.e., minuteamounts of material on the order of 1 to about 100,000 copies of rarecells or target rare molecules. Since this process involves traceanalysis at the detection limits of the mass spectrometers, these minuteamounts of material can only be detected when detection volumes areextremely low, for example, 10⁻¹⁵ liter, so that the concentrations arewithin the detection. Examples of methods and apparatus in accordancewith the principles described herein reduce or avoid evaporation.

Obtaining reproducibility in amounts of MS label or MS label precursorreleased for a rare cell or a target rare molecule requires measuringthe formation and essentially complete recovery of the carrier and labelparticles. Therefore, in one approach the carrier particles, labelparticles, linking group and/or MS label or MS label precursor may bemade fluorescent by virtue of the presence of a fluorescent moleculesuch as, but not limited to, FITC, rhodamine compounds, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde, fluorescent rare earthchelates, amino-coumarins, umbelliferones, oxazines, Texas red,acridones, perylenes, indacines such as, e.g.,4,4-difluoro-4-bora-3a,4a-diaza-s-indacene and variants thereof,9,10-bis-phenylethynylanthracene, squaraine dyes and fluorescamine, forexample. A fluorescent microscope may then be used to determine thelocation of the carrier particles, label particles, linking group,and/or MS label or MS label precursor before and after treatment. Thisserves as a confirmative measure of the system function and is valuedfor additional information on the location of the rare cell or targetrare molecule on the cellular structure or a capture particle.

Kits for Conducting Methods

The apparatuses and reagents of the invention may be present in a kituseful for conveniently performing the methods of the invention. In oneembodiment, a kit comprises a packaged combination of an essentiallynon-absorbent membrane and modified affinity agents, one for eachdifferent target rare molecule. The kit may also comprise one or moreunlabeled antibodies or nucleic acid probes directed at non-rare cellsso that they can be eliminated from analysis. Depending on whether themodified affinity agent comprises an MS label precursor or an alterationagent, the kit may also comprise the other of the MS label precursor orthe alteration agent that is not part of the modified affinity agent.The kit may also include a substrate for a moiety that reacts with an MSlabel precursor to generate an MS label. In addition, the kit may alsocomprise one or more of a fixation agent, a permeabilization agent, anda blocking agent to prevent non-specific binding to the cells, forexample. Other reagents for performing the method may also be includedin the kit, the nature of such reagents depending upon the particularformat to be employed. The reagents may each be in separate containersor various reagents can be combined in one or more containers dependingon the cross-reactivity and stability of the reagents. The kit canfurther include other separately packaged reagents for conducting themethod such as ancillary reagents, binders, containers for collection ofsamples, and supports for cells such as, for example, microscope slides,for conducting an analysis, for example.

The relative amounts of the various reagents in the kits can be variedwidely to provide for concentrations of the reagents that substantiallyoptimize the reactions that need to occur during the present methods andfurther to optimize substantially the sensitivity of the methods. Underappropriate circumstances one or more of the reagents in the kit can beprovided as a dry powder, usually lyophilized, including excipients,which on dissolution will provide for a reagent solution having theappropriate concentrations for performing a method in accordance withthe principles described herein. The kit can further include a writtendescription of a method utilizing reagents in accordance with theprinciples described herein.

The phrase “at least” as used herein means that the number of specifieditems may be equal to or greater than the number recited. The phrase“about” as used herein means that the number recited may differ by plusor minus 10%; for example, “about 5” means a range of 4.5 to 5.5.

The following examples further describe the specific embodiments of theinvention by way of illustration and not limitation and are intended todescribe and not to limit the scope of the invention. Parts andpercentages disclosed herein are by volume unless otherwise indicated.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

Various modifications of the invention and many further embodimentsthereof, in addition to those shown and described herein, will becomeapparent to those skilled in the art from the full contents of thisdocument, including references to the scientific and patent literaturecited herein. The subject matter herein contains important information,exemplification and guidance that can be adapted to the practice of thisinvention in its various embodiments and equivalents thereof.

EXAMPLES Example 1: Release of Liquid Droplets from Membrane

A total of three essentially non-absorbent membranes were employed fortesting. The first membrane was an essentially non-absorbent membranethat contained an 8×8 mm² silicon region consisting of 6400 microwellsapproximately 70 μm in diameter and 360 μm tall. The bottom of each wellwas covered by a 1 μm thick silicon nitride (Si₃N₄) rigid membrane witha 5 μm hole approximately centered within the well opening. The angleformed at the intersection of a surface of the membrane and the hole was90°. The second membrane was an essentially non-absorbent 1 μm thicksilicon nitride (Si₃N₄) rigid membrane of 8×8 mm² that consisted of asingle microwell with a region containing about 108,000 pores of 5 μmdiameter with the second membrane approximately centered within themicrowell opening. The angle formed at the intersection of a surface ofthe membrane and all pore holes was 90° and did not vary by more than1°. The third membrane was an essentially non-absorbent membrane ofpolycarbonate that was flexible. The third membrane was 3.7 cm² and waspositioned at the bottom of a single microwell. The third membrane hadabout 100,000 pores of 8 μm diameter. The angle formed at theintersection of a surface of the membrane and the hole of the porevaried from 30 to 150° between individual pores.

ESI occurs when the electric field strength at a solvent-air interfaceis ample in magnitude to overcome the forces due to surface tension ofthe liquid. At this point, the liquid is drawn into a cone from whichcharged droplets were expelled. These droplets underwent evaporation andfission cycles to ultimately produce gas-phase ions that were drawn intothe vacuum system of a mass spectrometer for analysis. In the generationof an electrospray directly from the membrane surface in this example,the solution to be sprayed must sufficiently wet the top-side of thechip (etched silicon wafer housing) which contains the microwells. Asolvent which displays ideal wettability with the surface willinherently fill the wells upon solvent addition, thus providing acapillary flow for continuous solvent delivery during spray events. Theback side of the Si₃N₄ membrane should ideally have a non-wettinginteraction with the spray solvent. This type of interaction isolatesthe liquid to single drops on each of the 5-μm pores. The presence ofindividual droplets creates a high degree of curvature (compared to aflat, wetted surface) which produces greater electric field strengthunder the application of an electric potential, thus aiding in theformation of an electrospray. Additionally, by positioning the capillaryinlet of a mass spectrometer in close proximity to the bottom side ofthe membrane, electric field strength is further enhanced and allows thegeneration of the electrospray from selected regions of the membrane,thus recovering spatial information. The spray solvent should besufficiently polar and have a surface tension low enough to permitelectrospray at electric field strengths lower than those which produceelectrical breakdown of air.

Acetonitrile and methanol were selected as the spray solvents forinitial tests of direct membrane spray. Experiments were performed inwhich the standard straight capillary for the atmospheric pressure inlet(API) of a THERMO LTQ (linear ion trap) mass spectrometer (from ThermoElectron North America LLC) was replaced with an extended capillarywhich was bent at a 90° angle, such that the opening was pointing up. Inall experiments, the membrane was positioned with the bottom sideparallel to the ground approximately 1 mm distant from the bentcapillary inlet. The bottom side of the membrane and the bent capillarywere illuminated using a diode laser and video was recorded with a CMOScamera.

In the first set of experiments, 50 μL of methanol (or acetonitrile) waspipetted directly onto the top side of the membrane and potential wasapplied by directing the plasma from an antistatic gun towards thesolvent. When potential was applied in this manner, discrete sprayevents were visualized and recorded mass spectra showed peaks typical ofspraying the same solution via nanoESl. Upon the depletion of solvent,spectra were drastically different and were characteristic of those seenwhen firing the antistatic gun unobstructed at the MS inlet.

Further experiments showed that a porous membrane will spray but theamount of material sprayed is variable (20% cv) and the time at whichthe ejection of analyte ions and charged droplets containing analyteoccurs is variable as well by more than 100 msec making the trapping ofions difficult as it occurs over short time scales and with small sprayvolumes; thus, resulting in a method that is not quantitative. As acontrol, an impermeable layer having pores of fixed orientation andbeing flexible (causing angle of greater than 1 degree) was tested. Theresult with this layer was that material sprayed varied by more than 20%CV. The membrane employed having characteristics in accordance with theprinciples described herein yielded spray amounts that were less than 1%CV.

A comparison of the performance of flexible and non-flexible membraneswas made by spraying a solution containing a quaternary peptide (FC-2)from both the flexible and non-flexible membranes. This comparison wasmade using membranes that had previously been used to filter blood. Inthe case of the flexible membrane, the membrane was positionedapproximately 0.5 mm above the bent MS inlet and a 6.5 kV potential wasapplied to a wire (the electric field generator) positionedapproximately 5 mm above the membrane. A solution (25 μL of 4:1methanol:water with 0.1% formic acid) containing the FC-2 peptide wasthen applied to the top of the membrane while recording MS/MS spectra ofthe ion m/z 412. When electrosprayed in positive mode, the FC-2 peptideproduces a molecular ion at m/z 412 that when subjected to CID fragmentsprimarily to m/z 353. In the case of the non-flexible membrane, theprocedure was identical with the following changes: the voltage of theelectric field generator was set to 6.0 kV and the amount of liquidapplied was 5 μL. The concentration of the FC-2 peptide in each case wasadjusted to 1, 10, and 100 nM. CID spectra of m/z 412 are shown in FIGS.8A-C and FIGS. 9A-C for the flexible and non-flexible membranes,respectively. Spray formation from the flexible membrane occurred over aperiod of 1-3 seconds after applying liquid to the membrane and aphotograph of the developed spray is shown in FIG. 10 . In the case ofthe non-flexible membrane, a comparable spray plume was not observed,suggesting that the spray formation occurs from individual wells.

1-10. (canceled)
 11. A method for detecting a target analyte from aheterogeneous sample, the method comprising: providing an apparatus thatincludes an essentially non-absorbent membrane comprising at least onepore, a microwell operably associated with the membrane, and an electricfield generator operably associated with the membrane introducing aheterogeneous sample to at least the membrane; introducing to the samplea plurality of affinity agents that each comprise a first molecule,wherein the plurality of affinity agents specifically bind the targetanalyte in the sample; removing unbound affinity agents; introducing oneor more additional molecules to the sample, wherein the one or moreadditional molecules interact with the first molecule to form a massspectrometry label; providing voltage to the sample via the electricfield generator to release a droplet through the at least one pore,wherein the droplet comprises a portion of the sample and the massspectrometry label; and analyzing the droplet for presence of the massspectrometry label, wherein the presence of the mass spectrometry labelindicates presence of the target analyte in the sample.
 12. The methodaccording to claim 11, wherein the first molecule is a mass spectrometrylabel precursor.
 13. The method according to claim 12, wherein the oneor more additional molecules is an alteration agent that interacts withthe mass spectrometry label precursor to form the mass spectrometrylabel.
 14. The method according to claim 11, wherein the first moleculeis an alteration agent.
 15. The method according to claim 12, whereinthe one or more additional molecules is a mass spectrometry precursorlabel that interacts with the alteration agent to form the massspectrometry label.
 16. The method according to claim 11, wherein thefirst molecule is a mass spectrometry label precursor, and the one ormore additional molecules are first and second alteration agents thatinteract with the mass spectrometry label precursor to form the massspectrometry label.
 17. The method according to claim 11, wherein theaffinity agent is a particulate.
 18. The method according to claim 11,wherein the affinity agent is a non-particulate.
 19. The methodaccording to claim 11, wherein the target analyte is a rare cell and theheterogeneous sample is a heterogeneous biological sample.
 20. Themethod according to claim 11, further comprising quantifying the targetanalyte in the sample by quantifying an amount of mass spectrometrylabel analyzed.
 21. A sample analysis method comprising: introducing asample suspected of comprising a target analyte to a membrane thatcomprises a pore; introducing one or more reagents to the sample on themembrane to generate a mass spectrometry label associated with targetanalyte if present in the sample. applying an electric field to themembrane to thereby generate one or more droplets of the sample that areexpelled from the pore of the membrane and are introduced into a massspectrometer; detecting via the mass spectrometer a presence of thetarget analyte by detecting a presence of the mass spectrometry label;extracting a portion of the sample associated with the pore of themembrane from the membrane if the target analyte is present based onresults from the detecting step; and analyzing the extracted portion ofthe sample.
 22. The method according to claim 21, wherein introducingone or more reagents to the sample on the membrane comprises:introducing to the sample a plurality of affinity agents that eachcomprise a first molecule, wherein the plurality of affinity agentsspecifically bind the target analyte in the sample; removing unboundaffinity agents; and introducing one or more additional molecules to thesample, wherein the one or more additional molecules interact with thefirst molecule to form a mass spectrometry label.
 23. The methodaccording to claim 22, wherein the first molecule is a mass spectrometrylabel precursor.
 24. The method according to claim 23, wherein the oneor more additional molecules is an alteration agent that interacts withthe mass spectrometry label precursor to form the mass spectrometrylabel.
 25. The method according to claim 22, wherein the first moleculeis an alteration agent.
 26. The method according to claim 25, whereinthe one or more additional molecules is a mass spectrometry precursorlabel that interacts with the alteration agent to form the massspectrometry label.
 27. The method according to claim 22, wherein thefirst molecule is a mass spectrometry label precursor, and the one ormore additional molecules are first and second alteration agents thatinteract with the mass spectrometry label precursor to form the massspectrometry label.
 28. The method according to claim 22, wherein theaffinity agent is a particulate.
 29. The method according to claim 22,wherein the affinity agent is a non-particulate.
 30. The methodaccording to claim 21, wherein the target analyte is a rare cell and thesample is a heterogeneous biological sample.
 31. The method according toclaim 21, further comprising quantifying the target analyte in thesample by quantifying an amount of mass spectrometry label analyzed.